Pegylated carfilzomib compounds

ABSTRACT

The present invention provides polymeric pegylated carfilzomib compounds, and pharmaceutically acceptable salts thereof, of Formula I 
     
       
         
         
             
             
         
       
     
     wherein R 1 , R 2 , linker, PEG, n and o are as defined herein. The invention also provides methods of making and using these compounds to treat cancer, and particularly to treat hematological malignancies including multiple myeloma.

FIELD OF THE INVENTION

The present invention relates to pegylated carfilzomib compounds,pharmaceutical compositions comprising the compounds, and methods anduses thereof for treating cancer, including hematologic malignanciessuch as multiple myeloma, and solid tumors.

BACKGROUND OF THE INVENTION

Cancer is one of the most widespread diseases and a leading cause ofdeath worldwide. In the United States alone, cancer is the secondleading cause of death, surpassed only by heart disease. Cancer is oftencharacterized by deregulation of normal cellular processes orunregulated cell proliferation.

Multiple myeloma (MM) is a progressive and malignant neoplastic type ofcancer originating from plasma cells. It is characterized by abnormalaccumulation of malignant plasma cells within bone marrow, and itaccounts for approximately 13% of all hematologic cancers (Palumbo andAnderson, 2011). In 2015, about 26,850 new cases were expected to bediagnosed with MM, and about 11,240 people were expected to die from thedisease in the United States (ACS, 2015). The incidence of MM hasincreased steadily due to increased life expectancy of the generalpopulation in the United States (Warren et al., 2013). The disease mostcommonly affects the elderly population, with the median age ofincidence around 69 years old (Howlander et al., 2013; ACS, 2015).

The therapeutic goals of management of MM are to provide symptomaticrelief, achieve disease control and provide prolonged remissions(Kurtin, 2013). Conventionally, a combination of high dosechemotherapeutic agents (melphalan, vincristine, cyclophosphamide,doxorubicin, liposomal doxorubicin, bendamamustine) followed byautologous stem-cell transplantation (ASCT) has been utilized to treatyoung, treatment-naïve and medically fit patients (less than 65 years ofage) (Palumbo et al., 2011). Age, comorbid conditions and geriatricassessment are the major criteria for deciding patients' eligibility totolerate high-dose therapy (HDT) followed by ASCT (Palumbo et al.,2014). For elderly patients ineligible for HDT and ASCT, melphalan plusprednisone had been the standard therapy for several decades (Palumbo etal., 2011; Rodriguez et al., 2012). During the last decade, treatmentalgorithm of MM underwent a paradigm change with the introduction ofnovel immunomodulatory agents (such as thalidomide, lenalidomide, andpomalidomide) and targeted proteasome inhibitors (bortezomib andcarfilzomib) (Richardson et al., 2007; Dmoszyńska, 2008; Gupta et al.,2013).

Carfilzomib is a tetrapeptide epoxy ketone proteosome inhibitor thatbinds selectively and irreversibly to the constitutive proteosome andimmunoproteosome. More specifically, the epoxyketone electrophilicwarhead binds to the catalytic threonine residue of the β5 subunit ofthe proteasome protein. CFZ is well tolerated with acceptable toxicityprofile. Carfilzomib, polymorphic forms, methods of making,formulations, its use and other carfilzomib attributes are described inUS20050245435, US20140105921 and PCT publications WO2006017842,WO2009045497, WO2014169897, WO2013169282, WO2014011695, WO2006063154,WO2014015016, and WO2010048298, each specification of which is herebyincorporated herein by reference in its entirety.

Carfilzomib has shown an encouraging response rate in patients withrelapsed and refractory MM and with newly diagnosed patients with MM. Tothis end, carfilzomib was first approved (as Kyprolis®) for treatment inpatients with relapsed and refractory MM in July 2012 as a single agenttherapy. More recently Kyprolis was approved in combination withlenalidomide and dexamethasone (July 2015) and in combination withdexamethasone (January 2016) for the treatment of patients with relapsedand refractory MM who have received one to three lines of therapy. Theapproved treatment regimen for carfilzomib is to administer it to thepatient by infusion, either over a short 10 minute period or over aslower, longer 30 minute duration of time. This infusion is to occur for2 consecutive days per week for three consecutive weeks in a 28 daycycle. Thus, to comply with this treatment schedule, patients need todrive or be driven two times per week on consecutive days to anauthorized drug administration center, such as a doctor's office, aclinic or a hospital, where carfilzomib can be properly and safelyadministered. This may be inconvenient or impractical, or may simply bea burden, to some patients, increasing the likelihood of reduced ordecreased compliance with, or even complete non-compliance of, the fulland complete course of the prescribed carfilzomib therapeutic regimen.

Carfilzomib is rapidly metabolized and cleared in humans. Carfilzomib, asmall tetrapeptide compound, exhibits a short half-life in-vivo of about60 minutes or less in humans. One mechanism of carfilzomib clearance isvia hepatic blood flow, resulting in the relatively brief half-life forcarfilzomib. Drug products possessing short half lives or rapidclearance in general tend to exhibit reduced target coverage leading todecreased and/or shortened biological inhibitory activity. To overcomesuch shortfalls, additional drug is typically administered to providemore drug and prolonged efficacy at the biological site of action.Hence, both the rapid clearance and the twice weekly frequency of dosingof carfilzomib leave room for possible improvements in efficacy,delivery and/or patient compliance.

Carfilzomib, as currently approved (Kyprolis®), is a sterile lyophilizedformulation comprising sulfabutylether beta cyclodextrin (SBECD) and asodium citrate buffer. The lyophilate is reconstituted with sterilewater, and infused or injected into the patient. The SBECD excipientacts primarily as a solubilizing additive for carfilzomib, and forms acomplex with carfilzomib thereby improving carfilzomib water solubility.

History has revealed that attempts to solve weaknesses of drug productshave led to the preparation of alternative forms of these medicinalcompounds, including production of pro-drug versions, in attempts toenhance their drug pK and/or PD properties. For instance, Greenwald etal disclose Prodrugs of Amine Containing Compounds (J. Med. Chem., 1999,42, 3657-3667). WO2005063777 discloses benzylphosphate and substitutedbenzylphosphate prodrugs for the treatment of pulmonary inflammation.WO20090152160 discloses inhaled carbaprotacyclin and prostacyclinprodrugs for the treatment of arterial hypertension. US patentpublication no. 20040100225 discloses acyloxymethyl pro-drugs ofimatinib (Gleevec®). Also, PCT publication WO2011084846 disclosesacyloxymethyl pro-drugs of risperidone. These pro-drug disclosures teachalkyl-acyloxymethyl linked pro-drugs. Another example, US patentapplication publication no. US20140105921 describes carfilzomib andother epoxyketone proteasome inhibitor pro-drugs having an acyloxymethyllinker connecting the inhibitor to polyethylene glycol units (PEG).However, these carfilzomib pro-drug compounds have been found to releasequinone methide byproducts during metabolism in vivo, which may bepotentially toxic and may present a safety risk. To this end, it wouldbe desirable to identify alternative forms of carfilzomib and/oralternative ways to deliver the active pharmaceutical ingredientcarfilzomb to patients while maintaining or possibly improving theefficacy and/or safety of the currently approved carfilzomib treatments.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides novel polymeric carfilzomib compounds,i.e., modified structures of carfilomib, that deliver therapeuticanti-cancer benefits to the patient while maintaining comparable orlonger carfilzomib plasma concentrations and proteasome exposure. Tothis end, these polymeric carfilzomib compounds provide proteosomalinhibitory activity comparable to that of the currently approvedcarfilzomib cyclodextrin IV formulation.

Particularly, the present invention provides pegylated carfilzomibcompounds, having improved water solubility, and which are useful totreat various types of cancer, including without limitation, multiplemyeloma. More particularly, the pegylated compounds provided herewithmaintain or exhibit suitable bioavailability and reduce or completelyeliminate the need for solubilizing excipients or agents such assulfobutylether-β-cyclodextrin. The present invention further provides amethod of preparing the pegylated carfilzomib compounds, pharmaceuticalcompositions comprising the same, and methods of using the compounds andcompositions for treating various forms of cancer such as multiplemyeloma.

In one aspect of the invention, the pegylated carfilzomib compoundsdescribed herein include one or more covelantly linked PEG moieties that(i) can confer enhanced solubility, permeability, pharmacokinetics (pK)and/or pharmacodynamics (PD) properties to carfilzomib when comparedwith the corresponding approved carfilzomib product that does notcontain such polymeric moieties; and (ii) can be cleaved or removed invivo after administration to a subject thereby further providing freecarfilzomib, that has proven safety and efficacy capabilities to treatvarious cancers, including without limitation, multiple myeloma.

The pegylated carfilzomib compounds provided by the present inventionfurther provide potential benefits including, without limitation,extended release allowing for reduced dosing frequency, lower Cmax and,consequenetly, possibly reduced side effects when compared with thepresently approved carfilzomib product. An improved safety profile ofthe compounds of the invention may also result from enhanced aqueoussolubility which could facilitate alternative modes of administration,such as for example subcutaneous administration, from the presentlyapproved infusion mode of administration. A modified pK and/orbiodistribution profile for the pegylated carfilzomib compounds of theinvention may result in improved efficacy in treating cancers, includingwithout limitation, multiple myeloma and solid tumors. Further, thepegylated carfilzomib compounds of the invention provide formulationoptions with potentially improved chemical and temperature stability,lower dosing volume and the potential to eliminate a lyophilizationstep, part of the manufacturing procedure for the currently approvedcarfilzomib drug product.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph of curves generated by rates of conversion of severalexemplified pegylated compounds of carfilzomib to free carfilzomib inhuman plasma (representative of human pK);

FIG. 2 is a graph reflecting the pK of representative pegylatedcompounds of carfilzomib in rat plasma;

FIG. 3 is a graphic illustration of the effects of carfilzomib andcompound Example 1 on chymotrypsin-like proteasome activity in theblood, adrenal gland, heart and liver tissues;

FIG. 4 is a graphic illustration of mean carfilzomib plasmaconcentrations over time for Examples 13, 26 and 34 of the invention;

FIG. 5 is a graph illustrating the efficacy of Example 13 versusCFZ-captisol formulation in a mouse xenograph cancer model;

FIG. 6 is a graphic illustration of the survival of the mice from theStudy illustrated in FIG. 5;

FIG. 7 is a graph illustrating a second efficacy study of Example 13versus CFZ-captisol formulation in a mouse xenograph cancer model;

FIG. 8 is a figure of the NMR spectrum for Example 23;

FIG. 9 is a figure of the cellular CT-L activity for carfilzomib;

FIG. 10 is a figure of the cellular CT-L activity for compound example5;

FIG. 11 is a figure of the cellular CT-L activity for compound example35;

FIG. 12 is a figure of the cellular CT-L activity for compound example36;

FIG. 13 is a figure of the cellular CT-L activity for compound example37;

FIG. 14 is a figure of the cellular CT-L activity for compound example38; and

FIG. 15 is a figure of the in-vivo CT-L activity for compound examples35 and 36.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel pegylated carfilzomib compounds,pharmaceutical compositions comprising the compounds, methods of makingthe compounds, and uses of the compounds and compositions including thecompounds for the treatment of cancer, including treatment ofhematologic malignancies such as multiple myeloma, lymphoma, leukemia,and treatment of other cancers such as solid tumors. Specifically, thepolymeric units of polyethylene glycol (PEG) linked carfilzomib possessvarious pharmacokinetic (pK) and/or pharmacodynamics (PD) propertiescomparable to or improved over that of the currently approved IVadministered Kyprolis® (carfilzomib).

Carfilzomib is an epoxy ketone protease inhibitor described in U.S. Pat.Nos. 7,417,042 and 7,737,112, among others. The pegylated carfilzomibcompounds described in the present invention (i) generally conferenhanced solubility, permeability, pK and/or PD properties relative tofree drug carfilzomib not containing such PEG moieties; and (ii) can becleaved in vivo thereby releasing the freely active drug carfilzomib. Inthe embodiments presented by the invention, the N-terminal “cap” of thecarflizomib (e.g., the morpholino cap) is converted to a quaternary salt(e.g., by the addition of a N-acyloxyphenylmethyl group). In someembodiments, the quaternary salt contains a PEG moiety. In someembodiments, the pegylated compounds are cleavable by pH change and/orenzymes such as, but not limited to, esterases, cytochrome P450,phosphodiesterase, phosphoamidase, phosphatase, and DT-diaphorase, orany combination thereof. In some embodiments, the PEG is a linear PEG Insome embodiments, the PEG is a bifunctional PEG that can conjugate 1-2compounds per PEG In some embodiments, the PEG is a four-arm PEG thatcan conjugate 1-4 compounds per PEG In some embodiments, the PEG is aneight-arm PEG with a hexaglycerin core that can conjugate 1-8 compoundsper PEG In some embodiments, the PEG is an eight-arm PEG with atripentaerythritol core that can conjugate 1-8 compounds per PEG In someembodiments, the PEG is a branched two-arm PEG In some embodiments, thePEG is a branched four-arm PEG In addition, the compounds can furtherinclude solubilizers, permeability enhancers, masking agents,macromolecular carriers, targeting moieties, and biologics to improvehalf-life and disease specificity that are attached directly to thecompound or indirectly attached via a spacer moiety.

The terms “aspect” and embodiment” are used interchangeably herein.

In aspect 1 of the invention, the invention provides a pegylatedcarfilzomib compound having a structure of formula I

or a pharmaceutically acceptable salt thereof, wherein

R¹ is C₁₋₁₀alkyl or C₃₋₇ cycloalkyl;

each R², independently, is C₁₋₆ alkyl, —OCH₃ or halogen;

o is an integer selected from 0, 1, 2 or 3;

linker is a moiety having the structure of

-   -   wherein R³ is H or CH₃;    -   n is an integer selected from 1, 2, 3 or 4;    -   p is an integer selected from 0, 1, 2, 3 or 4;    -   q is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8 or 9;    -   r is an integer selected from 0, 1, 2, 3, 4 or 5; and

PEG is a polyethylene glycol polymeric moiety having a molecular weightranging from about 500 to about 20,000.

In aspect 1a of the invention, the invention provides a pegylatedcarfilzomib compound having a structure of formula I

or a pharmaceutically acceptable salt thereof, wherein

R¹ is C₁₋₁₀alkyl or C₃₋₇ cycloalkyl;

each R², independently, is C₁₋₆ alkyl, —OCH₃ or halogen;

o is an integer selected from 0, 1, 2 or 3;

linker is a moiety having the structure of

wherein R³ is H or CH₃; and

p is an integer selected from 0, 1, 2, 3 or 4;

n is an integer selected from 1, 2, 3 or 4; and

PEG is a polyethylene glycol polymeric moiety having a molecular weightranging from about 500 to about 20,000.

In aspect 2 of the invention, the invention provides the pegylatedcarfilzomib compound of aspect 1 having a structure of Formula II

wherein

R¹ is C₁₋₁₀alkyl or C₃₋₇cycloalkyl;

R² is C₁₋₆alkyl, —OCH₃ or halogen;

linker is a moiety having the structure of

wherein R³ is H or CH₃;

n is an integer selected from 1, 2, 3 or 4;

p is an integer selected from 0, 1, 2, 3 or 4;

q is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8 or 9;

r is an integer selected from 0, 1, 2, 3, 4 or 5;

X is a counter ion salt selected from a chloride, a bisulfate, asulfate, a nitrate, a phosphate, an alky-sulfonate or an aryl-sulfonate;and

PEG is a polyethylene glycol polymeric moiety having a molecular weightranging from about 2000 to about 20,000.

In aspect 3 of the invention, the invention provides the compound ofaspects 1, 1a and 2 wherein R¹ is C₁₋₁₀alkyl.

In aspect 4 of the invention, the invention provides the compound of anyone of aspects 1, 1a, 2 and 3 wherein each R², independently, is H, CH₃or halogen.

In aspect 5 of the invention, the invention provides the compound of anyone of aspects 1, 1a, 2, 3 and 4 wherein each R², independently, is H,CH₃, Cl or F.

In aspect 5a of the invention, the invention provides the compound ofany one of aspects 1, 1a, 2, 3 and 4 wherein each R², independently, isH, CH₃ or F.

In aspect 6 of the invention, the invention provides the compound of anyone of aspects 1, 1a, 2, 3, 4 and 5 wherein the linker is a moietyhaving the structure of

wherein R³ is H or CH₃;

q is an integer selected from 1, 2, 3, 4 or 5; and

r is an integer selected from 0, 1, 2, 3 or 4.

In aspect 6a of the invention, the invention provides the compound ofany one of aspects 1, 1a, 2, 3, 4 and 5 wherein the linker is a moietyhaving the structure of

wherein R³ is H or CH₃.

In aspect 7 of the invention, the invention provides the compound of anyone of aspects 1, 1a, 2, 3, 4, 5 and 7 wherein the linker is

wherein R³ is H or CH₃;

-   -   q is 4; and    -   r is 2.

In aspect 7a of the invention, the invention provides the compound ofany one of aspects 1, 1a, 2, 3, 4, 5 and 7 wherein the linker is

wherein R³ is H or CH₃.

In aspect 8 of the invention, the invention provides the the compound ofany one of aspects 1, 1a, 2, 3, 4, 6, 6a, 7 and 7a wherein R³ is H.

In aspect 9 of the invention, the invention provides the compound of anyone of aspects 1-8 wherein R¹ is methyl, ethyl, propyl, isopropyl,butyl, t-butyl, pently, hexyl or heptyl.

In aspect 10 of the invention, the invention provides the compound ofany one of aspects 1-9 wherein R¹ is methyl, ethyl, propyl, isopropyl,butyl, t-butyl, pentyl, hexyl or heptyl; and the linker is

In aspect 10a of the invention, the invention provides the compound ofany one of aspects 1-9 wherein R¹ is methyl, ethyl, propyl, isopropyl,butyl, t-butyl, pentyl, hexyl or heptyl; and the linker is

It should be noted that in aspects 1, 1a, 2 and aspects 3-10 that theterm “or a pharmaceutically acceptable salt thereof” may include acounter ion salt of the quaternary nitrogen cationic charge, such asthat illustrated in formula II of aspect 2, or those illustrated inaspects 11-24 hereinbelow. Further, it should be noted that it isintended that the term “any one of aspects 1-X” also include allsub-aspects of 1-X disclosed herein, including without limitationsub-aspects 1a, 5a, 6a, 7a and 10a.

In aspect 11 of the invention, the invention provides a pegylatedcarfilzomib compound according to any one of aspects 1-10, having thestructure of

wherein R¹ is C₁₋₁₀alkyl;

R² is C₁₋₆alkyl, —OCH₃ or halogen;

R³ is H or CH₃;

X⁻ is a counter anion selected from chloride anion and a alkyl-sulfonateanion;

n is 4; and

PEG is a polyethylene glycol polymeric moiety having a molecular weightranging from about 2000 to about 20,000.

In aspect 12 of the invention, the invention provides the compound ofany one of aspects 1-11 wherein the compound is

wherein X is a halide, a sulfonate or an alkyl-sulfonate counterionsalt.

In aspect 12a of the invention, the invention provides the compound ofany one of aspects 1-11 wherein the compound is

wherein X is a halide, a sulfonate or an alkyl-sulfonate counterionsalt.

In aspect 13 of the invention, the invention provides the compound ofany one of aspects 1-11 wherein the compound is

wherein X is a halide, a sulfonate or an alkyl-sulfonate counterionsalt.

In aspect 14 of the invention, the invention provides the compound ofany one of aspects 1-11 wherein the compound is

In aspect 15 of the invention, the invention provides the compound ofany one of aspects 1 and 2 wherein R¹ is methyl, ethyl, propyl,isopropyl, butyl, t-butyl, pently, hexyl or heptyl;

each R², independently, is CH₃ or halogen;

linker is a moiety having the structure of

wherein R³ is H or CH₃; and

PEG is a polyethylene glycol polymeric moiety having a molecular weightof 2000, 3000, 5000 or 20,000.

In aspect 16 of the invention, the invention provides the compound ofaspect 15 wherein R¹ is methyl, ethyl, propyl, isopropyl, butyl,t-butyl, pently, hexyl or heptyl;

each R², independently, is CH₃;

linker is a moiety having the structure of

wherein R³ is H; and

PEG is a polyethylene glycol polymeric moiety having a molecular weightof 3000, 5000 or 20,000.

In aspect 17 of the invention, the invention provides the compound ofany one of aspects 1-16 wherein the compound is an individual compoundas represented in examples 1-34 described hereinbelow in Table 2, or apharmaceutically acceptable salt thereof.

In aspect 18 of the invention, the invention provides the compound ofany one of aspects 1-17 wherein the compound is

or a pharmaceutically acceptable salt thereof.

In aspect 19 of the invention, the invention provides the compound ofany one of aspects 1-18 wherein the compound is

In aspect 19a of the invention, the invention provides the compound ofany one of aspects 1-18 wherein the compound is

In aspect 20 of the invention, the invention provides the compound ofany one of aspects 1-18 wherein the compound is

In aspect 21 of the invention, the invention provides the compound ofany one of aspects 1-18 wherein the compound is

In aspect 22 of the invention, the invention provides the compound ofany one of aspects 1-18 wherein the compound is

In aspect 23 of the invention, the invention provides the compound ofany one of aspects 1-18 wherein the compound is

In aspect 24 of the invention, the invention provides the compound ofany one of aspects 1-18 wherein the compound is

In aspect 25 of the invention, the invention provides the compound ofany one of aspects 1-18 wherein the compound is

In aspect 26 of the invention, the invention provides the compound ofany one of aspects 1-18 wherein the compound is

In aspect 27 of the invention, the invention provides the compound ofany one of aspects 1-16 wherein the PEG has a weight ranging from about2K to about 20K.

In aspect 28 of the invention, the invention provides the compound ofany one of aspects 1-16 wherein the PEG has a weight of 3K, 5K or 20K.

In aspect 29 of the invention, the invention provides the compound ofany one of aspects 1-16 that is a pharmaceutically acceptable saltcomprising a counter anion selected from a chloride anion, a bisulfateanion, a sulfate anion, a nitrate anion, a phosphate anion, analky-sulfonate anion or an aryl-sulfonate anion.

In aspect 30 of the invention, the invention provides the compound ofaspect 29 wherein the counter anion is a chloride anion or analky-sulfonate anion.

In aspect 31 of the invention, the invention provides the compound ofaspect 29 wherein the counter anion is a chloride anion ormethane-sulfonate anion.

In aspect 32 of the invention, the invention provides a pharmaceuticalcomposition comprising the compound according to any one of aspects 1-26and a pharmaceutically acceptable excipient, carrier or diluent.

In aspect 33 of the invention, the invention provides pharmaceuticalcomposition of aspect 32 that is administered orally or parenterallyadministrable by infusion or injection.

In aspect 34 of the invention, the invention provides the pharmaceuticalcomposition according to any one of aspects 32-33 that comprises one ormore of the compounds according to any one of aspects 1-26 inconjunction with a pharmaceutically acceptable excipient, carrier ordiluent.

In aspect 35 of the invention, the invention provides the pharmaceuticalcomposition according to any one of aspects 32-34 that comprises atleast two compounds according to any one of aspects 1-28 in conjunctionwith a pharmaceutically acceptable excipient, carrier or diluent.

In aspect 36 of the invention, the invention provides a method oftreating multiple myeloma comprising administering to a patient in needthereof, a therapeutically effective amount of the compound of any oneof aspects 1-31 or the pharmaceutical composition of any one of aspects32-35.

In aspect 37 of the invention, the invention provides the method ofaspect 34 wherein the multiple myeloma is relapsed, refractory orrelapsed and refractory multiple myeloma.

In aspect 38 of the invention, the invention provides the method ofaspect 36 wherein the multiple myeloma is newly diagnosed multiplemyeloma.

In aspect 39 of the invention, the invention provides the a process ofmaking the compound according to any one of aspects 1-16, the processcomprising the step of

wherein X− is a counter ion salt selected from the group consisting of achloride anion, a bisulfate anion, a sulfate anion, a nitrate anion, aphosphate anion, an alky-sulfonate anion or an aryl-sulfonate anion, andPEG has a weight ranging from about 2K to about 20K, to prepare acompound of Formula I

In aspect 40 of the invention, the invention provides the a process ofmaking the compound of Formula I according to aspect 1, the processcomprising the step of

wherein

X− is a counterion salt of a chloride anion, a bisulfate anion, asulfate anion, a nitrate anion, a phosphate anion, an alky-sulfonateanion or an aryl-sulfonate anion;

PEG has a weight ranging from about 2K to about 20K; and R¹, R², R⁴ ando are as defined in aspect 1, to prepare a compound of Formula I.

In other aspects, the invention provides compounds having the formula(SM)_(m)-PEG, or a pharmaceutically acceptable salt thereof, in whicheach SM is an independently selected compound of formula I or of formulaII as defined in above and anywhere herein and is attached to PEG ofdefined weight at n number of locations, wherein n is 2-10 (e.g., n=4 inthe compound of aspect 14; See also Table I hereinbelow).

In some embodiments, the nitrogen atom of the carfilzomib morpholinering is substituted with the benzylic ester moiety as shown in formulasI and II, thereby forming a quaternary nitrogen atom and wherein thepositive charge associated with the quaternary nitrogen atom is balancedby a pharmaceutically acceptable anion, as defined herein by X⁻.

In some aspects of the invention, the compounds described hereinthemselves exhibit lower therapeutic activity when compared with saidcorresponding epoxy ketone protease inhibitors and exhibit enhancedsolubility, permeability, pharmacokinetics and/or pharmacodynamicsproperties in vivo when compared with said corresponding epoxy ketoneprotease inhibitors.

In some aspects of the invention, the polymeric moiety or peg moiety iscleaved from the carfilzomib active ingredient by pH change and/orenzymes such as, but not limited to, esterases, cytochrome P450,phosphodiesterase, phosphoamidase, phosphatase, and DT-diaphorase, orany combination thereof.

In some aspects of the invention, the PEG is a linear PEG In someaspects of the invention, the PEG is a bifunctional PEG that canconjugate 1-2 compounds per PEG. In some aspects of the invention, thePEG is a four-arm PEG that can conjugate 1-4 compounds per PEG In someaspects of the invention, the PEG is an eight-arm PEG with ahexaglycerin core that can conjugate 1-8 compounds per PEG In someaspects of the invention, the PEG is an eight-arm PEG with atripentaerythritol core that can conjugate 1-8 compounds per PEG In someaspects of the invention, the PEG is a branched two-arm PEG In someaspects of the invention, the PEG is a branched four-arm PEG Inaddition, the compounds can further include solubilizers, permeabilityenhancers, masking agents, macromolecular carriers, targeting moieties,and biologics to improve half-life and disease specificity that areattached directly to the compound or indirectly attached via a spacermoiety.

While not wishing to be bound by theory, it is possible that thecompounds of the invention may mask or partially mask the proteaseinhibitory activity temporarily until the pegylated linked moieties havebeen cleaved releasing free carfilzaomib, a tested and regulatoryapproved active pharmaceutical moiety, into the systemic circulation canreduce undesired side effects, which may otherwise be associated withvarious routes of administration. To this end, the pegylated carfilzomibcompounds of the present invention may act as pro-drugs of carfilzomib.Alternatively, as the pegylated moiety is positioned near the morpholineend of the carilzomib tetrapeptide backbone structure, the compounds ofthe present invention, prior to peg cleavage, may very well possessactive proteasome inhibitory activity.

The beneficial properties of the compounds of the present invention mayalso facilitate subcutaneous administration and extend half-life ofcarfilzomib, e.g., to beyond four hours. In aspect 41 of the invention,the human plasma half-life of the compounds of the invention is longerthan 0.5 hr. In aspect 42 of the invention, the human plasma half-lifeof the compounds is between 0.5 and 5 hr. In aspect 43 of the invention,the human plasma half-life of the compound is longer than 5 hr. Inaspect 44 of the invention, the human plasma half-life of the compoundis between 5 and 100 hr. In aspect 45 of the invention, the human plasmahalf-life of the compound is longer than 100 hr. In aspect 46 of theinvention, the human plasma half-life of the compound is between 100 and836 hr. In aspect 47 of the invention, the human plasma half-life of thecompound is between 200 and 300 hr. In aspect 48 of the invention, thehuman plasma half-life of the compound is about 267 hr. In aspect 49 ofthe invention, the human plasma half-life of the compound is up to 836hr. By virtue of extending the half-life of carfilzomib, the inventionpotentially improves dosing as well as patient convenience andcompliance in treatment with carfilzomib.

In aspect 50 of the invention, the invention provides a pharmaceuticalcomposition, which includes a PEG carfilzomib compound as describedanywhere herein and a pharmaceutically acceptable excipient, carrier ordiluent.

In other aspects or embodiments of the invention, which are laterdescribed herein, methods are featured for treating a disease orcondition selected from the group consisting of cancer, autoimmunedisease, graft or transplant-related condition, neurode generativedisease, fibrotic-associated condition, ischemic-related conditions,infection (viral, parasitic or prokaryotic) and diseases associated withbone loss, the method includes administering to a patient atherapeutically effective amount of a compound as described anywhereherein. In still further aspects, methods for treating cancer (e.g.,multiple myeloma, e.g., multiple myeloma that is relapsed and/orrefractory) in a patient are provided by the invention, which includeadministering to a patient a therapeutically effective amount of acompound as described herein.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Methods and materials aredescribed herein for use in the present disclosure; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety, as if here written. In case of conflict,the present specification, including definitions, will control. Otherfeatures and advantages of the disclosure will be apparent from thefollowing detailed description and from the claims.

As used herein, the term “aspect” is used synonymously with the term“embodiment.”

Definitions

The following definitions should further assist in understanding theterms as used herein and the scope of the invention described herein.

The term “C_(x-y)alkyl” refers to substituted or unsubstituted saturatedhydrocarbon groups, including straight-chain alkyl and branched-chainalkyl groups that contain from x to y carbons in the chain. The term“haloalkyl” refers to alkyl groups in which at least one hydrogen atomis replace by a halo (e.g., fluoro, chloro, bromo, iodo), e.g., CH₂F,CHF₂, trifluoromethyl and 2,2,2-trifluoroethyl.

The terms “C_(2-y)alkenyl” and “C_(2-y)alkynyl” refer to substituted orunsubstituted unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but that contain atleast one double or triple bond, respectively. In some embodiments,divalent groups alkenylene and alkynylene include from 2 to 12 carbonatoms. In certain embodiments, alkylene and alkynylene include from 2 to10 carbon atoms. In certain embodiments, alkylene and alkynylene includefrom 2 to 6 carbon atoms (e.g., 2, 3, 4, 5, or 6 carbon atoms).

The term “alkoxyl” refers to an alkyl group having an oxygen attachedthereto. Representative alkoxyl groups include methoxy, ethoxy, propoxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxy.

The term “C_(3-y)cycloalkyl”, as used herein, refers to a fullysaturated, substituted or unsubstituted, ring in which each atom of thering is carbon, and the ring contains from 3 to γ carbon atoms in size.For instance, the term C₃₋₇ cycloalkyl is intended to mean a carbocyclicring containing anywhere from 3 to 7 carbon atoms in size. Such ringsinclude, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyland cycloheptyl rings. These rings may further be substituted asspecified.

The terms “cancer” and “cancerous” when used herein refer to or describethe physiological condition in subjects that is typically characterizedby unregulated cell growth. Examples of cancer include, withoutlimitation, hematologic malignancies or blood borne cancers such asmultiple myeloma and leukemia, and other cancers such as carcinoma,lymphoma, sarcoma, and blastoma. More particular examples of suchcancers include squamous cell carcinoma, lung cancer, pancreatic cancer,cervical cancer, bladder cancer, hepatoma, breast cancer, coloncarcinoma, and head and neck cancer. While the term “cancer” as usedherein is not limited to any one specific form of the disease, it isbelieved that the methods of the invention will be particularlyeffective for cancers, in a subject, which have become resistant in somedegree to treatment with anti-cancer agents, including withoutlimitation chemotherapeutic agents, antimitotic agents, anthracyclinesand the like, and for cancers which have relapsed post treatment withsuch anti-cancer agents.

The term “comprising” is meant to be open ended, including the indicatedcomponent(s) but not excluding other elements.

The term or abbreviation “eg” or “eg.” as used herein is intended tomean “example.”

The term “inhibitor” is meant to describe a compound that blocks orreduces an activity of an enzyme or system of enzymes, receptors, orother pharmacological target (for example, inhibition of proteolyticcleavage of standard fluorogenic peptide substrates such assuc-LLVY-AMC, Box-LLR-AMC and Z-LLE-AMC, inhibition of various catalyticactivities of the 20S proteasome). An inhibitor can act withcompetitive, uncompetitive, or noncompetitive inhibition. An inhibitorcan bind reversibly or irreversibly, and therefore the term includescompounds that are suicide substrates of an enzyme. An inhibitor canmodify one or more sites on or near the active site of the enzyme, or itcan cause a conformational change elsewhere on the enzyme. The terminhibitor is used more broadly herein than scientific literature so asto also encompass other classes of pharmacologically or therapeuticallyuseful agents, such as agonists, antagonists, stimulants, co-factors,and the like.

The terms “drug resistant” and “multidrug resistant” when used hereinrefers to cancer cells that have developed and/or are resistant to drug.These include cancer cells exhibiting little to no efficacy or decreasedefficacy from that exhibited at the initial dose of the drug. The cancercells may be resistant to one drug or to multiple drugs of differentchemical structures that are directed to act at different biologicaltargets within the cancer cell.

The term “pharmaceutically-acceptable salt” embraces salts commonly usedto form alkali metal salts and to form addition salts of free acids orfree bases. The nature of the salt is not critical, provided that it ispharmaceutically-acceptable. Suitable pharmaceutically-acceptable acidaddition salts of the compound may be prepared from an inorganic acid orfrom an organic acid. Examples of such inorganic acids include, withoutlimitation, hydrochloric, hydrobromic, hydroiodic, nitric, carbonic,sulfuric and phosphoric acid. Examples of organic acids include, withoutlimitation, aliphatic, cycloaliphatic, aromatic, arylaliphatic,heterocyclic, carboxylic and sulfonic classes of organic acids, examplesof which are formic, acetic, adipic, butyric, propionic, succinic,glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic,glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic,anthranilic, mesylic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic(pamoic), methanesulfonic, ethanesulfonic, ethanedisulfonic,benzenesulfonic, pantothenic, 2-hydroxyethanesulfonic, toluenesulfonic,sulfanilic, cyclohexylaminosulfonic, camphoric, camphorsulfonic,digluconic, cyclopentanepropionic, dodecylsulfonic, glucoheptanoic,glycerophosphonic, heptanoic, hexanoic, 2-hydroxy-ethanesulfonic,nicotinic, 2-naphthalenesulfonic, oxalic, palmoic, pectinic,persulfuric, 2-phenylpropionic, picric, pivalic propionic, succinic,tartaric, thiocyanic, mesylic, undecanoic, stearic, algenic,β-hydroxybutyric, salicylic, galactaric and galacturonic acid.

Suitable pharmaceutically-acceptable base addition salts of the compoundinclude, without limitation, metallic salts such as salts made fromaluminum, calcium, lithium, magnesium, potassium, sodium and zinc, orsalts made from organic bases including primary, secondary, tertiaryamines and substituted amines including cyclic amines such as caffeine,arginine, diethylamine, N-ethyl piperidine, histidine, glucamine,isopropylamine, lysine, morpholine, N-ethyl morpholine, piperazine,piperidine, triethylamine, trimethylamine. All of the salts contemplatedherein may be prepared by conventional means from the correspondingcompound by reacting, for example, the appropriate acid or base with thecompound.

The term “proteasome” as used herein is meant to include immuno- andconstitutive proteasomes.

The term “refractory” when used here is intended to refer tonot-yielding to, resistant or non-responsive to treatment, stimuli(therapy) or cure, including resistance to multiple therapeutic curativeagents. “Refractory” when used herein in the context of characterizing acancer or tumor is intended to refer to the cancer or tumor beingnon-responsive or having a resistant or diminished response to treatmentwith one or more anticancer agents. The treatment typically iscontinual, prolonged and/or repetitive over a period of time resultingin the cancer or tumor relapsing or developing resistance or becomingrefractory to that very same treatment.

The term “subject” as used herein refers to any mammal, includinghumans, and animals such as cows, horses, dogs and cats. Thus, theinvention may be used in human patients as well as in veterinariansubjects and patients. In one embodiment of the invention, the compoundsof the invention may be administered to a human subject.

The phrase “therapeutically-effective” or “therapeutically effectiveamount” is intended to quantify the amount of the compound of theinvention, which when administered as part of a desired dosage regimen(to a patient, e.g., a human) alleviates a symptom, ameliorates acondition, or slows the onset of disease conditions according toclinically acceptable standards for the disorder or condition to betreated or the cosmetic purpose, e.g., at a reasonable benefit/riskratio applicable to any medical treatment. Thus, it is the amount of thecompound of the invention that can treat cancer, whether it is multiplemyeloma or other hematologic malignancy or a solid tumor.

The terms “treat”, “treating” and “treatment” as used herein refer totherapy, including without limitation, curative therapy, prophylactictherapy, and preventative therapy and generally include reversing,reducing, or arresting the symptoms, clinical signs, and underlyingpathology of a condition in manner to improve or stabilize a patient'scondition. Prophylactic treatment generally constitutes eitherpreventing the onset of disorders altogether or delaying the onset of apre-clinically evident stage of disorders in individuals. The term“prophylactic or therapeutic” treatment is art-recognized and includesadministration to the host of one or more of the subject compositions.If it is administered prior to clinical manifestation of the unwantedcondition (e.g., disease or other unwanted state of the host animal), orafter the condition has subsided, then the treatment is prophylactic,(i.e., it protects the host against developing the unwanted condition),whereas if it is administered after manifestation of the unwantedcondition, the treatment is therapeutic, (i.e., it is intended todiminish, ameliorate, or stabilize the existing unwanted condition orside effects thereof).

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more non-hydrogen atoms of the molecule. It will beunderstood that “substitution” or “substituted with” includes theimplicit proviso that such substitution is in accordance with permittedvalence of the substituted atom and the substituent, and that thesubstitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc. As used herein, the term “substituted” iscontemplated to include all permissible substituents of organiccompounds. In a broad aspect, the permissible substituents includeacyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and non-aromatic substituents of organiccompounds. The permissible substituents can be one or more and the sameor different for appropriate organic compounds. For purposes of thisdisclosure, the heteroatoms such as nitrogen may have hydrogensubstituents and/or any permissible substituents of organic compoundsdescribed herein which satisfy the valences of the heteroatoms.Substituents can include, for example, a halogen, a hydroxyl, a carbonyl(such as a carboxyl, an alkoxycarbonyl, a formyl, or an acyl), athiocarbonyl (such as a thioester, a thioacetate, or a thioformate), analkoxyl, a phosphoryl, a phosphate, a phosphonate, a phosphinate, anamino, an amido, an amidine, an imine, a cyano, a nitro, an azido, asulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, asulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic orheteroaromatic moiety. It will be understood by those skilled in the artthat the moieties substituted on the hydrocarbon chain can themselves besubstituted, if appropriate.

The term PEG, as used herein, is intended to have its commonlyunderstood and traditional meaning. Particularly, PEG is a moiety madeup of repeating poly(ethylene glycol) polymeric units, the precisenumber of which determines its molecule weight. For instance, the PEGmoiety used in the present invention can have any one of thearchitectures as described herein as well as, e.g., depicted in formulasA-I as shown in Table I. The unit of this molecular weight is daltons.Thus, it is intended that reference to a molecular weight of PEG as usedherein (the specification, claims and abstract), for example, referenceof “2K”, “3K”, “5K” and “20K” or “2000”, “3000”, “5000” or “20000” withrespect to a given PEG means a 2000 dalton (or 2 kilodalton), 3000dalton (or 3 kilodalton), 5000 dalton (or 5 kilodalton), and 20000dalton (or 20 kilodalton), respectively, PEG weight. Further, as usedherein “KDa” means kilodalton. It will be understood that thecarfilzomib compounds shown in Formulas A, B, C, D, E, F, H and I inTable I illustrate different PEG moieties covalently attached throughlinkers as described and defined by the invention herein. To assist withunderstanding each Formula in Table I,

wherein R¹ and R² are as defined herein in Formulas I and II. Counteranion X⁻ is as defined in Formulas I and II herein. For instance,counter anion X⁻ can be Cl⁻, HSO₄ ⁻, SO₄ ⁻², NO₃ ⁻, H₂PO₄ ⁻,alkyl/aryl-SO₃ ⁻ such as a tosylate (tosylsulfonic acid), mesylate(methanesulfonic acid) or benzylate (benzylsulfonic acid) anion. The PEGportion of the carfilzomib compound typically has a molecular weight inthe rage from about 400 daltons to about 50,000 daltons. The linkerportion of the compounds illustrated in Formulas A-I below are also asdefined herein in Formulas I and II. For example, the linker may be

wherein R³ is H or Me.

TABLE I Carfilzomib Entry Structure PEG Type molecules A

linear 1 B

bifunctional 2 C

4-Arm 4 D

8-Arm Hexaglycerine Core 8 E

8-Arm Tripentaerythritol Core 8 F

Branched 2-Arm 1 G

Branched 3-Arm 1 H

Branched 4-Arm 1 I

Branched 4-Arm 1

In embodiment 51, PEG has a molecular weight of greater than 1 kDa.

In embodiment 52, PEG has a molecular weight of about 1 kDa.

In embodiment 53, PEG has a molecular weight of greater than 2 kDa.

In embodiment 54, PEG has a molecular weight of about 2 kDa.

In embodiment 55, PEG has a molecular weight of greater than 5 kDa.

In embodiment 56, PEG has a molecular weight of about 5 kDa.

In embodiment 57, PEG has a molecular weight of greater than 10 kDa.

In embodiment 58, PEG has a molecular weight of about 10 kDa.

In embodiment 59, PEG has a molecular weight of greater than 20 kDa.

In embodiment 60, PEG has a molecular weight of about 20 kDa.

In embodiment 61, PEG has a molecular weight of greater than 30 kDa.

In embodiment 62, PEG has a molecular weight of about 30 kDa.

In embodiment 63, PEG has a molecular weight of greater than 40 kDa.

In embodiment 64, PEG has a molecular weight of about 40 kDa.

In embodiment 65, PEG has a molecular weight of greater than 50 kDa.

In embodiment 66, prior to conjugation to epoxy ketone proteaseinhibitors, PEG has a plurality of reactive functional groups (e.g.,azide groups).

In some embodiments, prior to conjugation to epoxy ketone proteaseinhibitors, PEG has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 reactive functionalgroups (e.g., azide groups).

The PEG carfilzomib compounds of the invention possess a polyethyleneglycol (PEG) polymer chain conjugated to the carfilzomib activepharmaceutical ingredient (API). To this end, the invention provides inaspect 67, a carfilzomib PEG compound of formula I or formula II whereinn is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or10. In embodiment 68, n is 8. In embodiment 69, n is 4. In embodiment70, n is 2. In embodiment 71, n is 1

General Synthesis and Representative Examples of the Invention

As described, the pegylated carfilzomib compounds in formulas I and IIare cleavable polymer PEG carriers of the active pharmaceuticalingredient, carfilzomib (Formulas I and II) and release free carfilzomibin vivo. The solubilizing polymer carrier, polyethylene glycol (PEG) ofdesired size and/or weight are commercially available and may be, forexample, purchased from ThermoFisher Scientific, Sigma-Aldrich andsimilar commercial suppliers of polymeric materials. The PEG group maybe appended to carfilzomib as a quaternary salt on the morpholine ringin a variety of linkers, including via a self-immolativepara-alkanoyloxy substituted benzyl linker, as described herein. Thelinker contains a latent nucleophilic phenol group which becomeselectron-donating after a biological or chemical triggering mechanismand then initiates an electronic cascade leading ultimately to therelease of carfilzomib. The unique combination of solubilizing polymercarrier and quaternary salt formation results in conjugates withextraordinarily high aqueous solubility. Carfilzomib is released fromthe conjugate enzymatically via an esterase enzyme or chemically by ahydroxide catalyzed hydrolysis. It should be noted that this PEGconjugate alone/itself is active as a proteasome inhibitor, and quicklyreleases the more active pharmaceutical ingredient carfilzomib when theconjugate is exposed to an appropriate esterase enzyme or to a slightlybasic environment. The rate of displacement may be varied over a timerange by the introduction of sterically bulky group(s) which limitenzyme access, and/or electron density modulating group(s) at positionR² in formulas I and II.

Abbreviations: The following abbreviations used throughout both thegeneral schemes and the examples, are intended to mean the following:

DCM dichloromethane; methylene dichloride

DMF dimethylformamide

DMSO dimethyl sulfoxide

EtOAc ethyl acetate

MeOH methanol

mpk milligram per kilogram; mg/kg

RT, rt room temperature

NaCl sodium chloride

tBuOH t-butanol; t-butyl alcohol

The starting material carfilzomib, used to prepare the compounds of theinvention, are described in PCT publications WO2006017842, WO2009045497,WO2014169897, WO2013169282, WO2014011695, WO2006063154, WO2014015016,WO2010048298 and U.S. Pat. Nos. 7,714,042 and 7,737,112, eachspecification of which is hereby incorporated herein by reference in itsentirety.

Enzymatic and/or chemical hydrolysis of the phenyl ester provides acarboxylic acid (II) and a phenolate intermediate (I) which undergoesrapid 1, 6 elimination to provide free carfilzomib and a quinone methidewhich remains covalently attached to the solubilizing PEG polymer.Quinone methides are known to be reactive Michael acceptors and arebelieved to present risks related to potential genotoxicity. In thisinvention, permanent attachment of the quinone methide linker byproductto the polymer may attenuate toxicity by preventing cellular access andlowering reactivity to serum nucleophiles. The most likely fate ofintermediate III in vivo is reaction with water to form a benzylalcohol-polymer adduct that is quickly removed from the body byexcretion.

Scheme 2 illustrates one metabolic pathway for the carfilzomib polymercompounds described in WO2014011695. Here, as illustrated above, thecarfilzomib-polymer conjugates interact with an esterase enzyme or aresubject to chemical attack as shown by the arrow. This attack results inthe release of a free quinone methide (encapsulated above) upon esterhydrolysis. This methide intermediate is free to react further withcellular nucleophiles, which may possibly result in toxicity. Thepegylated carfilzomib compounds of the present invention avoid thispotentially toxic byproduct, as described in scheme 1.

The carfilzomib-PEG compounds provided by the present invention areprepared in a two-step procedure, as shown in Scheme 3. Carfilzomib isfirst reacted with an appropriately substituted para-alkanoyloxysubstituted benzyl halide (1) to give a quaternary salt intermediate(2). The quaternary salt bromide or iodide anion may be exchanged to apharmaceutically acceptable anion such as bisulfate, sulfate, nitrate,dihydrogen phosphate or alkyl/aryl sulfonate via ion exchange resin togive intermediate (3). This intermediate is conveniently appended with areactive group suitable for reaction with a complimentarilyfunctionalized polymer reagent (4) to afford desired product 5. A largenumber of PEG reagents are commercially available in a range ofmolecular weights, architectures, end group chemistries, and number ofreactive end groups (arms) (see Table 1). They may be directlycompatible with the linker chemistries described in this disclosure ormay require some further chemical manipulation by known methods.Branched chain and multi-arm PEGs may offer advantages over linear PEGssuch as the potential for higher drug loading, improved stability,and/or lower formulation viscosities.

Scheme 4 illustrates “Click” chemistries, such as Huisgen 1, 3-dipolarazide/alkyne cycloaddition and aminooxy/aldehyde oximation that areparticularly well suited for polymer and polymeric PEG attachments dueto high chemical yields, inoffensive byproducts, large thermodynamicdriving forces, and starting material availability.

Huisgen 1, 3-dipolar azide/alkyne cycloaddition requires that the benzylgroup be substituted with an alkyne group (A1-(1-6)) capable of reactingwith an azide functionalized polymeric carrier such as PEG-Azide (—N₃)to give a 1,2,3-triazole linked conjugate (A4-(1-6)). The alkyne moietymay be directly linked or linked via an alkyl spacer (A1-1), linked viaan ether (A1-2,3), thioether, sulfoxide or sulfone (A1-4) bond, orlinked via an amide bond (A1-5,6). Many azido substituted PEG reagentsare now commercially available in a wide variety of sizes andarchitectures, but also may be readily prepared from any availablePEG-alcohol via activation by mesylation or tosylation followed byreaction with an azide salt. The cycloaddition reaction can be performedusing commercially available cuprous salt catalysts, but works moreefficiently using a mixture of copper (II) (e.g. copper (II) sulfate,copper (II) methanesulfonate) and a reducing agent (e.g. sodiumascorbate) to produce Cu (I) in situ. Since copper (I) is unstable inaqueous solution and in the presence of oxygen, stabilizing ligands suchas tris-(benzyltriazolylmethyl)amine (TBTA),tris(3-hydroxypropyltriazolylmethyl)amine (THPTA),2-[4-({bis[(1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl]amino}methyl)-1H-1,2,3-triazol-1-yl]ethylhydrogen sulfate (BTTES) or2-[4-({bis[(1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl]amino}methyl)-1H-1,2,3-triazol-1-yl]aceticacid (BTTAA) may be optionally added. The reaction can be run at RT orat an elevated temperature in a variety of solvents, and mixtures ofwater and a variety of miscible organic solvents including alcohols,DMSO, DMF, tBuOH and acetone. The final PEG-carfilzomib product(A4-(1-6)) may be conveniently worked up by dilution of the reactionmixture with water or brine, extraction with an organic solvent such asDCM, and reprecipitation from isopropanol or ether/isopropanol mixturesuntil product of desired purity is obtained. The exposure ofintermediates or products to anions during workup procedures, such aschloride anions in brine, typically result in a mixture of anions in thefinal product, and a final anion exchange resin treatment may benecessary to ensure product salt homogeneity.

The intermediate quaternary halide salt (bromide or iodide, (A2-(1-6))can be converted to an anion which does not precipitate with the copper(I) catalyst such as methanesulfonate, bisulfate or sulfate (A3-(1-6))to achieve high reaction yields. In addition it may be desirable toexchange the halide anion to prevent opening of the epoxide and possibleformation of bromohydrin or iodohydrin side-products.

Synthesis of 4-(Bromomethyl)-2-(prop-2-ynyloxy)phenyl acetate(intermediate A1-2 in scheme 4) Step 1:4-Hydroxy-3-(prop-2-ynyloxy)benzaldehyde (1)

To a mixture of NaOtBu in DMF (150 mL) was added3,4-dihydroxybenzaldehyde (10 g, 72.5 mmol) in DMF (50 mL) at 20° C. Themixture was cooled with an ice bath and stirred while 3-bromoprop-1-yne(8.62 g, 72.5 mmol) was added portionwise, attempting to keep internaltemperature between 15-20° C. The reaction mixture was stirred at RT for2 hours. The mixture was diluted with water (300 mL) and extracted withEtOAc (200 mL×3). The combined organic layers were washed with water toremove DMF, dried over anhydrous Na₂SO₄, and concentrated to a brownsolid. The residue was crystallized repeatedly from DCM/Petroleum Ether(30 mL/500 mL) to afford compound 1. 1H NMR (CDCl3, 300 MHz,): δ 9.87(s, 1H), 7.54 (d, J=1.2 Hz, 1H), 7.49 (dd, J1=1.5 Hz, J2=8.1 Hz, 1H),7.09 (d, J=8.1 Hz, 1H), 4.82 (m, 2H), 2.62 (m, 1H).

Step 2: 4-Formyl-2-(prop-2-ynyloxy)phenyl acetate (2)

To a solution of compound 1 (10.00 g, 56.82 mmol) in DCM (150 mL) wasadded Et₃N (11.48 g, 113.64 mmol) followed by acetyl chloride (5.35 g,68.18 mmol) at 0° C. The reaction mixture was stirred at RT for 2 hours.The mixture was washed with saturated 2N aqueous HCl (100 mL) and water(50 mL), dried over anhydrous MgSO₄, and concentrated to afford compound2, which was used in the next step without further purification. ¹H NMR(CDCl₃, 400 MHz): δ 9.96 (s, 1H), 7.63 (d, J=1.6 Hz, 1H), 7.54 (dd,J₁=1.6 Hz, J₂=8.0 Hz, 1H), 7.25 (d, J=8.0 Hz, 1H), 4.79 (d, J=2.4 Hz,2H), 2.57 (t, J=2.4 Hz, 1H), 2.35 (s, 3H).

Step 3: 4-(Hydroxymethyl)-2-(prop-2-ynyloxy)phenyl acetate (3)

To a solution of compound 2 (12.00 g, 55.05 mmol) in DCM/MeOH (150 mL/15mL) was added NaBH₄ (3.06 g, 82.57 mmol) in small portions at 0° C. Thereaction mixture was stirred at RT for 30 min. The mixture was quenchedby acetone (5 mL), and concentrated. The residue was purified by flashcolumn chromatography on silica gel (Petroleum Ether/EtOAc=2:1) toafford compound 3. ¹H NMR (CDCl₃, 400 MHz): δ 7.16 (d, J=1.6 Hz, 1H),7.04 (d, J=8.0 Hz, 1H), 6.98 (dd, J₁=1.6 Hz, J2=8.0 Hz, 1H), 4.72 (d,J=2.4 Hz, 2H), 4.69 (s, 2H), 2.53 (t, J=2.4 Hz, 1H), 2.32 (s, 3H).

Step 4: 4-(Bromomethyl)-2-(prop-2-ynyloxy)phenyl acetate (4)

To a solution of compound 3 (11.50 g, 52.27 mmol) in DCM (150 mL) wereadded PPh₃ (20.50 g, 78.41 mmol) and NBS (11.04 g, 62.73 mmol) at 0° C.The reaction mixture was stirred at room temperature for 0.5 hour. Anexcess of solvent was concentrated and the residue was purified by flashcolumn chromatography on silica gel (Petroleum Ether/EtOAc=20:1) toafford compound 4 (7.82 g, 53% yield). ¹H NMR (CDCl₃, 400 MHz): δ 7.14(m, 1H), 7.02 (m, 2H), 4.73 (d, J=2.4 Hz, 2H), 4.48 (s, 2H), 2.55 (t,J=2.4 Hz, 1H), 2.32 (s, 3H).

Ion exchange may be accomplished by reaction of the intermediatequaternary halide with a silver salt or more practically, passagethrough an ion exchange resin, as shown in Scheme 5. The carfilzomibquaternary salt anion present in intermediates or final products may beefficiently converted to a different strong acid anion such asbisulfate, sulfate, dihydrogen phosphate, nitrate or alkyl/arylsulfonate via anion exchange resin. An anion exchange resin such asAmberlyst A26 (OH⁻ form) is pretreated with the desired acid or ammoniumsalt, and then the quaternary halide salt is passed through. Conjugatesprepared from weak acid anions such as acetate, formate or lactate areunstable due to the increased basicity of the quaternary salt andincompatibility with the ester trigger group.

Alternatively, the benzyl group (B1-(1-6)) may be substituted with acarbonyl (aldehyde or ketone) group which is capable of reacting with anaminooxy functionalized polymeric carrier such as PEG-aminooxy (—ONH₂)to provide a stable oxime linked conjugate (B4-(1-6)). The carbonylmoiety may be directly linked or linked via an alkyl spacer (B1-1),linked via an ether (B1-2,3), thioether, sulfoxide or sulfone (B1-4)bond, or linked via an amide bond (B1-5,6). Carfilzomib and benzylhalide (B1-(1-6)) are allowed to react at RT or at an elevatedtemperature in a suitable organic solvent such as acetonitrile toprovide quaternary intermediate (B2-(1-6)) as a bromide or iodide salt.It is desirable to exchange this halide anion to prevent opening of theepoxide and possible formation of bromohydrin or iodohydrinside-products. Anion exchange may be accomplished by reaction of theintermediate quaternary halide with a silver salt or more practically,passage through an ion exchange resin as described previously (Scheme5). The carfilzomib quaternary salt intermediate (B3-(1-6)) and thePEG-ONH₃ ⁺Y⁻ polymer reagent are then allowed to react at RT or at anelevated temperature in a suitable organic such as DCM or a mixedaqueous organic solvent. Oximation catalysts such as aniline,p-phenylenediamine, or 5-methoxyanthranilic acid may be optionally addedbut are not usually necessary. Note that the carfilzomib quaternary saltintermediate (B3-(1-6)) and PEG-aminooxy reagent anion salts areidentical to obviate the formation of a mixed anion salt final productand the need for any further anion manipulation. The finalPEG-carfilzomib product may be conveniently worked up by evaporation ofthe reaction solvent and re-precipitation of the residue fromisopropanol or ether/isopropanol mixtures until product of desiredpurity is obtained.

PEG-Aminooxy reagents may be commercially available or readily preparedfrom mesyl or tosyl activated PEG-Alcohols, PEG-Halides (A) or PEG-Amine(B) starting materials, as depicted in Scheme 7. Thetert-butyloxycarbonyl protected intermediate may be deprotected with astrong acid such as hydrogen chloride, methanesulfonic acid,trifluoroacetic acid, or sulfuric acid to give the PEG-Aminooxy reagentas a chloride, trifluoroacetate or sulfate salt. The PEG-Aminooxyreagent anion may be optionally exchanged for a different anion viaanion exchange resin.

It is readily understood by persons of ordinary skill in the art thatoximes may exist as two geometric isomers: a syn (Z)-isomer and an anti(E)-isomer, as depicted in Scheme 8. Many of the examples in thisdisclosure are aromatic aldoximes and exist only as (E)-isomers.Non-aromatic aldoximes and ketoximes can usually be completely separatedand obtained as a (Z)-isomer and an (E)-isomer. The pegylatednon-aromatic aldoximes and ketoximes described in this invention mayexist as separate (Z) and (E)-isomers or as a mixture of (Z) and(E)-isomers.

Alternatively, the carfilzomib-polymer conjugates described in thisinvention may be prepared in a one-step reaction of carfilzomib and apara-alkanoyloxy substituted benzyl halide pre-appended with the desiredpolymer chain, as shown in Scheme 9. The polymer chain may be appendedvia a wide variety of known chemistries or the alkyne/azide orcarbonyl/aminooxy chemistries described previously. This route may beless desirable due to the difficulty in separating PEG containingproducts from unreacted pegylated starting materials.

Representative Examples of the Invention

The following pegylated carfilzomib compounds are representativeexamples of the invention and are not intended to be construed aslimiting the scope of the present invention. The pegylated carfilzomibcompounds were prepared using the following two general PEG linkingmethods (A and B).

PEG Triazole-Linker Method A

Carfilzomib quaternary salt intermediate A3-(1-6) (1.5 eq), PEG-Azide (1eq) and (L)-ascorbic acid (0.75 eq) were mixed in DMF (50 mL/mmolPEG-Azide) to give a cream-colored suspension. The mixture was stirredvigorously for 5 minutes and a solution of copper (II) sulfatepentahydrate (0.3 eq) in water (10 mL/mmol PEG-Azide) added rapidlydropwise. The reaction darkened immediately to a yellowish-brown colorand the suspension turned clear within 5 min. After 1 hour, a secondportion of ascorbic acid (0.75 eq) was added and the reaction mixturestirred for 60 minutes. A third portion of ascorbic acid (0.38 eq) wasadded and the reaction mixture stirred overnight at RT. Water (100mL/mmol PEG-Azide) and NaCl (15 g/mmol PEG-Azide) were added and themixture stirred until the NaCl dissolved. The product was extracted withDCM (3×35 mL/mmol PEG-Azide). The extract was dried over anhydroussodium sulfate, filtered, and concentrated under vacuum at 40° C. Theresidue was dissolved in isopropanol (125 mL/mmol PEG-Azide) at 40° C.Once the solids dissolved completely, diethyl ether (90 mL/mmolPEG-Azide) was added and the solution cooled in an ice bath. Theresulting solid was filtered and the filter cake washed with 2-propanoland diethyl ether each twice. The filter cake was dissolved in DCM andconcentrated under vacuum. The residue was dissolved in warm (40° C.)isopropanol (200 mL/mmol PEG-Azide) and then allowed to cool in an icebath. The resulting solid was filtered and the filter cake washed with2-propanol and diethyl ether each twice and then dried under vacuum.

PEG Oxime-Linker Method B

Carfilzomib quaternary salt intermediate B3-(1-6) (1 eq), PEG-ONH₃ ⁺MsO⁻(0.8 eq) and 5-methoxyanthranilic acid (oximation catalyst, 0.3 eq) inDCM (15 mL/mmol B3-(1-6)) were stirred at RT until complete consumptionof the PEG reagent was observed by HPLC (ELS detector). The reactionmixture was evaporated to dryness and the residue dissolved inisopropanol (15 mL/mmol B3-(1-6)) at 40° C. The clear solution wascooled to RT and ether (5 mL/mmol B3-(1-6)) added to inducecrystallization. The mixture was cooled in an ice bath for 5-10 minutesand the formed solid collected by filtration. Recrystallization fromisopropanol/ether was repeated one or two more times until all theunreacted carfilzomib quaternary salt intermediate B3-(1-6) was removedas detected by HPLC. The final solid was dried under vacuum at 30° C.Typical yields: 60-80%; Typical reaction times: 10-30 min forintermediates containing an aldehyde function, 24 h for intermediateswith a ketone function.

Examples of PEG-Carfilzomib compounds prepared, PEG architecture and PEGlinker methodology are listed in Table 2. Table 2 further includes thesize and weight (Daltons) of the PEG adduct and method used to appendthe PEG moiety to the carfilzomib backbone.

TABLE 2 Example Structure PEG Size/Arms PEG Linker Method  1

20K/4 A  2

 5K/1 A  3

 5K/1 A  4

 5K/1 A  5

 5K/1 A  6

 5K/1 A  7

 5K/1 A  8

 5K/1 A  9

 5K/1 A 10

 5K/1 A 11

20K/4 A 12

20K/4 A 13

20K/4 A 14

20K/4 A 15

 5K/1 A 16

 5K/1 B 17

 5K/1 B 18

 5K/1 B 19

 5K/1 B 20

 5K/1 B 21

 2K/1 B 22

 2K/1 B 23

 3K/1 B 24

 3K/1 B 25

20K/4 B 26

 5K/1 B 27

 5K/1 B 28

 5K/1 B 29

20K/4 B 30

20K/4 B 31

 5K/1 B 32

 5K/1 B 33

 5K/1 B 34

 3K/1 A 35

 3K/1 A 36

 2K/1 A 37

 3K/1 A 38

 2K/1 A

Example 1:4-(4-Acetoxy-3-((1-(PEG_(20K))-4-Arm)-1H-1,2,3-triazol-4-yl)methoxy)benzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholin-4-iummethanesulfonate (7)

4-Hydroxy-3-(prop-2-ynyloxy)benzaldehyde (1)

To a mixture of NaH in DMSO (300 mL) was added 3,4-dihydroxybenzaldehyde(30 g, 217.39 mmol) in DMSO (50 mL) at 20° C. The mixture was stirredfor 30 min and 3-bromoprop-1-yne (25.87 g, 217.39 mmol) was added. Thereaction mixture was stirred at RT for one hour. The mixture was pouredinto ice water (800 mL) and the resulting solution was adjusted to pH=2.The mixture was extracted with EtOAc (500 mL×3), dried over anhydrousMgSO₄, and concentrated. The residue was crystallized repeatedly fromDCM/Petroleum Ether (30 mL/500 mL) to afford compound 1 (30 g, 78%yield); ¹H NMR (CDCl₃, 300 MHz,): δ 9.87 (s, 1H), 7.54 (d, J=1.2 Hz,1H), 7.49 (dd, J₁=1.5 Hz, J₂=8.1 Hz, 1H), 7.09 (d, J=8.1 Hz, 1H), 4.82(m, 2H), 2.62 (m, 1H).

4-Formyl-2-(prop-2-ynyloxy)phenyl acetate (2)

To a solution of compound 1 (10.00 g, 56.82 mmol) in DCM (150 mL) wasadded Et₃N (11.48 g, 113.64 mmol) followed by acetyl chloride (5.35 g,68.18 mmol) at 0° C. The reaction mixture was stirred at RT for 2 hours.The mixture was washed with saturated 2 N aqueous HCl (100 mL) and water(50 mL), dried over anhydrous MgSO₄, and concentrated to afford compound2 (12 g, 97% yield), which was used in the next step without furtherpurification; ¹H NMR (CDCl₃, 400 MHz): δ9.96 (s, 1H), 7.63 (d, J=1.6 Hz,1H), 7.54 (dd, J₁=1.6 Hz, J₂=8.0 Hz, 1H), 7.25 (d, J=8.0 Hz, 1H), 4.79(d, J=2.4 Hz, 2H), 2.57 (t, J=2.4 Hz, 1H), 2.35 (s, 3H)

4-(Hydroxymethyl)-2-(prop-2-ynyloxy)phenyl acetate (3)

To a solution of compound 2 (12.00 g, 55.05 mmol) in DCM/MeOH (150 mL/15mL) was added NaBH₄ (3.06 g, 82.57 mmol) in small portions at 0° C. Thereaction mixture was stirred at RT for 30 min. The mixture was quenchedby acetone (5 mL), and concentrated. The residue was purified by flashcolumn chromatography on silica gel (Petroleum Ether/EtOAc=2:1) toafford compound 3 (10.32 g, 85% yield); ¹H NMR (CDCl₃, 400 MHz): δ 7.16(d, J=1.6 Hz, 1H), 7.04 (d, J=8.0 Hz, 1H), 6.98 (dd, J₁=1.6 Hz, J2=8.0Hz, 1H), 4.72 (d, J=2.4 Hz, 2H), 4.69 (s, 2H), 2.53 (t, J=2.4 Hz, 1H),2.32 (s, 3H).

4-(Bromomethyl)-2-(prop-2-ynyloxy)phenyl acetate (4)

To a solution of compound 3 (11.50 g, 52.27 mmol) in DCM (150 mL) wereadded PPh₃ (20.50 g, 78.41 mmol) and NBS (11.04 g, 62.73 mmol) at 0° C.The reaction mixture was stirred at RT for 0.5 hour. An excess ofsolvent was concentrated and the residue was purified by flash columnchromatography on silica gel (Petroleum Ether/EtOAc=20:1) to affordcompound 4 (7.82 g, 53% yield); ¹H NMR (CDCl₃, 400 MHz): δ 7.14 (m, 1H),7.02 (m, 2H), 4.73 (d, J=2.4 Hz, 2H), 4.48 (s, 2H), 2.55 (t, J=2.4 Hz,1H), 2.32 (s, 3H).

4-(4-Acetoxy-3-(prop-2-yn-1-yloxy)benzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholin-4-iummethanesulfonate (6)

To a solution of compound 4 (5.85 g, 20.67 mmol) in MeCN (50 mL) wasadded(S)-4-methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide(4.96 g, 6.89 mmol). The reaction mixture was stirred at 45° C. for 2days. An excess of solvent was concentrated and the residue was purifiedby flash column chromatography on silica gel (EtOAc/MeOH=100:6) toafford desired compound 5, which was transformed into the correspondingmesylate (3.2 g, 50% yield) by treatment with ion exchange resin; ¹H NMR(CDCl₃, 400 MHz): δ 9.66 (m, 1H), 7.83 (m, 1H), 7.36 (m, 1H), 7.26-7.14(m, 13H), 6.72 (m, 1H), 5.18 (m, 1H), 4.90 (m, 2H), 4.77 (m, 2H),4.53-4.36 (m, 4H), 4.26 (m, 3H), 4.08 (m, 1H), 3.92 (m, 2H), 3.74 (m,1H), 3.46 (m, 1H), 3.35 (m, 1H), 3.13 (m, 1H), 3.04 (m, 2H), 2.81 (s,3H), 2.75 (m, 2H), 2.62 (m, 1H), 2.33 (m, 3H), 2.20 (m, 1H), 2.12 (m,1H), 1.70-1.53 (m, 3H), 1.50-1.33 (m, 5H), 1.25 (m, 2H), 0.90-0.81 (m,12H).

Example 1 was prepared from compound 6 and PEG_(20K)(N₃)₄ followinggeneral pegylation procedure A; ¹H NMR (500 MHz, relaxation time=10 sec)DMSO-d₆ NMR: δ 9.50 (s, 4H), 8.50 (s, 4H), 8.39 (d, J=8 Hz, 4H), 8.27(d, J=8 Hz, 4H), 8.11 (s, 4H), 8.05 (d, J=8 Hz, 4H), 7.58 (s, 4H),7.26-7.29 (m, 12H), 7.12-7.23 (m, 32H), 7.03-7.06 (m, 4H), 5.26 (m, 8H),4.89-5.00 (m, 8H), 4.52-4.56 (m, 12H), 4.28-4.38 (m, 16H), 4.17-4.20 (m,4H), 4.05 (m, 16H), 3.81 (t, J=5.5 Hz, 8H), 3.63-3.66 (m, 8H), 3.50 (s,2098H), 3.10 (d, J=5 Hz, 4H), 2.94-2.98 (m, 12H), 2.72-2.78 (m, 4H),2.50-2.65 (m, 8H), 2.24 (s, 12H), 1.90-1.98 (m, 4H), 1.78-1.88 (m, 4H),1.58-1.66 (m, 8H), 1.39 (s, 12H), 1.25-1.38 (m, 12H), 0.836-0.882 (m,24H), 0.786-0.817 (m, 24H); Loading: 87%.

Example 2:4-(4-Acetoxy-2-((1-PEG_(5K)-1H-1,2,3-triazol-4-yl)methoxy)benzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholin-4-iummethanesulfonate (9)

2-Hydroxy-4-(methoxymethoxy)benzaldehyde (1)

To a solution of compound 2,4-dihydroxybenzaldehyde (5.04 g, 36.24 mmol)in THF (100 mL) were added DIPEA (6.52 g, 54.35 mmol) andchloro(methoxy)methane (3.21 g, 39.86 mmol). The reaction mixture wasstirred at RT overnight. An excess of solvent was concentrated and theresidue was purified by flash column chromatography on silica gel(Petroleum Ether/EtOAc=15:1) to afford compound 1 (3.96 g, 60% yield);¹H NMR (300 MHz, CDCl₃): δ 11.41 (s, 1H), 9.76 (s, 1H), 7.48 (dd, J1=2.7Hz, J2=8.4 Hz, 1H), 6.67 (dd, J1=2.4 Hz, J2=8.7 Hz, 1H), 6.62 (d, J=2.1Hz, 1H), 5.25 (d, J=2.7 Hz, 2H), 3.51 (d, J=3.0 Hz, 3H).

4-(Methoxymethoxy)-2-(prop-2-ynyloxy)benzaldehyde (2)

To a mixture of NaH (900 mg, 21.252 mmol) in DMSO (100 mL) was addedcompound 1 (2.0 g, 10.63 mmol) in DMSO (50 mL) at 20° C. The mixture wasstirred at the same temperature for 30 min and then 3-bromoprop-1-yne(1.90 g, 15.94 mmol) was added dropwise. The reaction mixture wasstirred at the same temperature for 4 hours and then was poured into icewater (100 mL). The resulting solution was adjusted to pH=2-3 and EtOAc(100 mL) was added. The two phases were separated and the water phasewas extracted with EtOAc (100 mL×3). The organic combined organic phaseswere dried and concentrated. The residue was purified by flash columnchromatography on silica gel (Petroleum Ether/EtOAc=3:1) to affordcompound 2 (1.89 g, 80% yield); ¹H NMR (300 MHz, CDCl₃): δ 10.34 (s,1H), 7.85 (d, J=9.3 Hz, 1H), 6.76 (m, 2H), 5.26 (s, 2H), 4.83 (d, J=2.4Hz, 2H), 3.52 (s, 3H), 2.60 (q, J=2.4 Hz, 1H).

4-Hydroxy-2-(prop-2-ynyloxy)benzaldehyde (3)

To a solution of compound 2 (5.1 g, 23.18 mmol) in propan-2-ol (100 mL)was added CBr₄ (760 mg, 2.32 mmol). The reaction mixture was refluxedovernight. An excess of solvent was concentrated and the residue waspurified by flash column chromatography on silica gel (PetroleumEther/EtOAc=3:1) to afford compound 3 (2.44 g, 60% yield); ¹H NMR (400MHz, DMSO-d6): δ 10.76 (s, 1H), 10.11 (s, 1H), 7.60 (d, J=8.8 Hz, 1H),6.59 (d, J=2.0 Hz, 1H), 6.52 (dd, J1=2.0 Hz, J2=8.8 Hz, 1H), 4.92 (d,J=2.4 Hz, 2H), 3.70 (q, J=2.4 Hz, 1H).

4-(Hydroxymethyl)-3-(prop-2-ynyloxy)phenol (4)

To a solution of compound 3 (2.45 g, 13.92 mmol) in MeOH (40 mL) wasadded NaBH₄ (618 mg, 16.698 mmol) in small portions at 0° C. Thereaction mixture was stirred at the same temperature for 1 hour and thenquenched with water (1.5 mL). An excess of solvent was concentrated andthe residue was re-dissolved in EtOAc (100 mL). The resulting solutionwas dried and concentrated to afford compound 4 (1.80 g, 74% yield),which was used in the next step without further purification; ¹H NMR(400 MHz, DMSO-d6): δ 6.98 (d, J=8.0 Hz, 1H), 6.32 (s, 1H), 6.26 (d,J=8.0 Hz, 1H), 4.65 (d, J=2.0 Hz, 2H), 4.32 (s, 2H), 3.54 (m, 1H).

4-(Hydroxymethyl)-3-(prop-2-ynyloxy)phenyl acetate (5)

To a solution of compound 4 (1.20 g, 6.74 mmol) in DCM (30 mL) was addedTEA (1.70 g, 16.85 mmol) followed by acetyl chloride (634 mg, 8 mmol)dropwise at 0° C. The reaction mixture was stirred at RT for 30 min. Anexcess of solvent was concentrated and the residue was purified by flashcolumn chromatography on silica gel (Petroleum Ether/EtOAc=5:1) toafford compound 5 (360 mg, 30% yield); ¹H NMR (400 MHz, DMSO-d6): δ 7.38(d, J=8.0 Hz, 1H), 6.79 (d, J=2.0 Hz, 1H), 6.75 (dd, J1=2.0 Hz, J2=8.0Hz, 1H), 5.09 (m, J=5.6 Hz, 1H), 4.82 (d, J=2.4 Hz, 1H), 4.46 (d, J=5.6HZ, 2H), 3.60 (q, J=2.4 Hz, 1H), 2.26 (s, 3H).

4-(Bromomethyl)-3-(prop-2-ynyloxy)phenyl acetate (6)

To a solution of compound 5 (360 mg, 1.64 mmol) in DCM (15 mL) was addedPPh₃ (515 mg, 1.96 mmol) followed by NBS (318 mg, 1.80 mmol) in smallportions at 0° C. The reaction mixture was stirred at the sametemperature for 30 min. An excess of solvent was concentrated and theresidue was purified by flash column chromatography on silica gel(Petroleum Ether/EtOAc=50:1) to afford compound 6 (190 mg, 41% yield);¹H NMR (400 MHz, CDCl3): δ 7.36 (d, J=8.4 Hz, 1H), 6.78 (d, J=2.0 Hz,1H), 6.73 (dd, J1=2.0 Hz, J2=8.4 Hz, 1H), 4.77 (d, J=2.4 Hz, 2H), 4.54(s, 2H), 2.56 (q, J=2.4 Hz, 1H), 2.31 (s, 3H).

4-(4-Acetoxy-2-(prop-2-yn-1-yloxy)benzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholin-4-iummethanesulfonate (8)

To a solution of compound 6 (190 mg, 0.67 mmol) in MeCN (10 mL) wasadded(S)-4-methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide(480 mg, 0.67 mmol). The reaction mixture was stirred at 45° C.overnight. An excess of solvent was concentrated and the residue waspurified by flash column chromatography on silica gel (MeOH/EtOAc=1:50)to afford desired compound 7, which was transformed into thecorresponding mesylate (340 mg, 74% yield) by treatment with ionexchange resin; ¹H NMR (400 MHz, CDCl₃): δ 9.68 (m, 1H), 7.88 (m, 1H),7.63 (m, 1H), 7.33˜7.16 (m, 10H), 6.89 (m, 3H), 6.50 (m, 1H), 5.16 (m,1H), 5.05 (m, 1H), 4.87 (m, 1H), 4.75 (m, 2H), 4.47 (m, 2H), 4.45˜4.12(m, 8H), 4.02 (m, 3H), 3.72 (m, 1H), 3.54 (m, 1H), 3.38 (m, 1H), 3.20(m, 1H), 3.06 (m, 2H), 2.80 (s, 3H), 2.74 (m, 2H), 2.63 (m, 2H),2.40˜2.08 (m, 5H), 1.64 (m, 2H), 1.47 (s, 3H), 0.85 (m, 12H).

The compound of Example 2 was prepared from compound 8 and PEG_(5K)N₃following general pegylation procedure A

Example 3:4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(2-((1-PEG_(5K)-1H-1,2,3-triazol-4-yl)methoxy)-4-(propionyloxy)benzyl)morpholin-4-iummethanesulfonate (9)

2-Hydroxy-4-(methoxymethoxy)benzaldehyde (1)

To a solution of 2,4-dihydroxybenzaldehyde (5.0 g, 36.23 mmol) in THFwere added DIPEA (6.52 g, 54.35 mmol) and chloro(methoxy)methane (3.21g, 39.86 mmol). The reaction mixture was stirred overnight. An excess ofsolvent was concentrated and the residue was purified by flash columnchromatography on silica gel (Petroleum Ether/EtOAc=15:1) to affordcompound 1 (3.96 g, 60% yield); ¹H NMR (300 MHz, CDCl₃): δ 11.41 (s,1H), 9.76 (s, 1H), 7.48 (dd, J1=2.7 Hz, J2=8.4 Hz, 1H), 6.67 (dd, J1=2.4Hz, J2=8.7 Hz, 1H), 6.62 (d, J=2.1 Hz, 1H), 5.25 (d, J=2.7 Hz, 2H), 3.51(d, J=3.0 Hz, 3H).

4-(Methoxymethoxy)-2-(prop-2-ynyloxy)benzaldehyde (2)

To a mixture of NaH (900 mg, 21.252 mmol) in DMSO (100 mL) was addedcompound 1 (2.0 g, 10.626 mmol) in DMSO (50 mL) at 20° C. The mixturewas stirred at the same temperature for 30 min and then3-bromoprop-1-yne (1.90 g, 15.94 mmol) was added dropwise. The reactionmixture was stirred at the same temperature for 4 hours and then waspoured into ice water (100 mL). The resulting solution was adjusted topH=2-3 and EtOAc (100 mL) was added. The two phases were separated andthe water phase was extracted with EtOAc (100 mL×3). The combinedorganic phases were dried and concentrated. The residue was purified byflash column chromatography on silica gel (Petroleum Ether/EtOAc=3:1) toafford compound 2 (1.89 g, 80% yield); ¹H NMR (300 MHz, CDCl₃): δ 10.34(s, 1H), 7.85 (d, J=9.3 Hz, 1H), 6.76 (m, 2H), 5.26 (s, 2H), 4.83 (d,J=2.4 Hz, 2H), 3.52 (s, 3H), 2.60 (q, J=2.4 Hz, 1H).

4-Hydroxy-2-(prop-2-ynyloxy)benzaldehyde (3)

To a solution of compound 2 (5.1 g, 23.18 mmol) in propan-2-ol (100 mL)was added CBr₄ (760 mg, 2.318 mmol). The reaction mixture was refluxedovernight. An excess of solvent was concentrated and the residue waspurified by flash column chromatography on silica gel (PetroleumEther/EtOAc=3:1) to afford compound 3 (2.44 g, 60% yield); ¹H NMR (400MHz, DMSO-d6): δ10.76 (s, 1H), 10.11 (s, 1H), 7.60 (d, J=8.8 Hz, 1H),6.59 (d, J=2.0 Hz, 1H), 6.52 (dd, J1=2.0 Hz, J2=8.8 Hz, 1H), 4.92 (d,J=2.4 Hz, 2H), 3.70 (q, J=2.4 Hz, 1H).

4-(Hydroxymethyl)-3-(prop-2-ynyloxy)phenol (4)

To a solution of compound 3 (2.45 g, 13.92 mmol) in MeOH (40 mL) wasadded NaBH₄ (618 mg, 16.698 mmol) in small portions at 0° C. Thereaction mixture was stirred at the same temperature for 1 hour and thenwas quenched by water (1.5 mL). An excess of solvent was concentratedand the residue was re-dissolved in EtOAc (100 mL). The resultingsolution was dried and concentrated to afford compound 4 (1.80 g, 74%yield), which was used in the next step without further purification; ¹HNMR (400 MHz, DMSO-d6): δ 6.98 (d, J=8.0 Hz, 1H), 6.32 (s, 1H), 6.26 (d,J=8.0 Hz, 1H), 4.65 (d, J=2.0 Hz, 2H), 4.32 (s, 2H), 3.54 (m, 1H).

4-(Hydroxymethyl)-3-(prop-2-ynyloxy)phenyl propionate (5)

To a solution of compound 4 (2.4 g, 13.5 mmol) in DCM/THF (30 mL/5 mL)were added Et₃N (3.41 g, 33.75 mmol) and propionic anhydride (1.93 g,14.8 mmol) at 0° C. The reaction mixture was stirred at RT overnight. Anexcess of solvent was concentrated and the residue was purified by flashcolumn chromatography on silica gel (Petroleum Ether/EtOAc=3:1) toafford compound 5 (850 mg, 30% yield); ¹H NMR (400 MHz, DMSO-d6): δ 7.38(d, J=8.0 Hz, 1H), 6.81 (d, J=2.4 Hz, 1H), 6.74 (dd, J1=2.4 Hz, J2=8.4Hz, 1H), 5.08 (br, s, 1H), 4.80 (d, J=2.4 Hz, 1H), 4.46 (s, 2H), 3.60(m, 1H), 2.20 (m, 2H), 1.16 (m, 3H).

4-(Bromomethyl)-3-(prop-2-ynyloxy)phenyl propionate (6)

To a solution of compound 5 (850 mg, 3.63 mmol) in DCM (40 mL) wereadded PPh₃ (1.24 g, 4.72 mmol) and NBS (767.4 mg, 4.36 mmol) at RT. Thereaction mixture was stirred for 30 min. An excess of solvent wasconcentrated and the residue was purified by flash column chromatographyon silica gel (Petroleum Ether/EtOAc=3:1) to afford compound 6 (780 mg,73% yield); ¹H NMR (400 MHz, CDCl₃): δ 7.34 (d, J=8.0 Hz, 1H), 6.78 (d,J=2.0 Hz, 1H), 6.73 (dd, J1=2.4 Hz, J2=8.4 Hz, 1H), 4.77 (d, J=2.4 Hz,1H), 4.54 (m, 1H), 2.60 (m, 2H), 2.56 (m, 1H), 1.27 (q, J=7.6 Hz, 3H).

4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(2-(prop-2-yn-1-yloxy)-4-(propionyloxy)benzyl)morpholin-4-iummethanesulfonate (8)

To a solution of compound 6 (780 mg, 2.626 mmol) in MeCN (10 mL) wasadded(S)-4-methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide(945 mg, 1.313 mmol). The reaction mixture was stirred at RT overnight.An excess of solvent was concentrated and the residue was purified byflash column chromatography on silica gel (EtOAc/MeOH=100:3) to affordthe desired compound 7 (760 mg, 57% yield), which was transformed intothe corresponding mesylate (740 mg, 97% yield) by treatment with ionexchange resin; ¹H NMR (400 MHz, CDCl₃): δ 9.64 (m, 1H), 7.88 (m, 1H),7.63 (m, 1H), 7.26 (m, 10H), 6.92 (m, 1H), 6.89 (m, 2H), 6.50 (m, 1H),5.16 (m, 1H), 5.05 (m, 1H), 4.87 (m, 1H), 4.78 (m, 2H), 4.47 (m, 2H),4.45˜4.12 (m, 8H), 3.72 (m, 1H), 3.54 (m, 1H), 3.38 (m, 1H), 3.20 (m,1H), 3.06 (m, 2H), 2.84 (m, 1H), 2.80 (s, 3H), 2.74 (m, 2H), 2.63 (m,3H), 2.40˜2.08 (m, 5H), 1.64 (m, 2H), 1.47 (s, 3H), 1.24 (m, 3H), 0.85(m, 12H).

The pegylated carfilzomib compound Example 3 was prepared from compound8 and PEG_(5K)N₃ following general pegylation procedure A.

Example 4:4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(4-(isobutyryloxy)-3-(((1-PEG_(5K)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)benzyl)morpholin-4-iummethanesulfonate (9)

tert-Butyl 5-formyl-2-hydroxybenzoate (1)

To a solution of compound 5-formyl-2-hydroxybenzoic acid (2.01 g, 12mmol) in 2-methylpropan-2-ol (70 mL) was added DCC (2.3 g, 12 mmol) atRT. The reaction mixture was stirred under reflux for 3 hours. Thesolvent was concentrated and the residue was purified by flash columnchromatography on silica gel (Petroleum Ether/EtOAc=5:1) to affordcompound 1 (1.8 g, 75% yield). ¹H NMR (300 MHz, CDCl₃): δ 11.76 (s, 1H),9.91 (s, 1H), 8.33 (d, J=2.1 Hz, 1H), 8.00 (dd, J₁=1.8 Hz, J2=8.7 Hz,1H), 7.11 (d, J=8.4 Hz, 1H), 1.68 (s, 9H).

tert-Butyl 5-formyl-2-(isobutyryloxy)benzoate (2)

To a solution of compound 1 (1.01 g, 4.5 mmol) in THF (20 mL) were addedpyridine (1.07 g, 13.5 mmol) and isobutyric anhydride (1.423 g, 9 mmol)at RT. The reaction mixture was stirred for 3 hours. An excess ofsolvent was concentrated and the residue was purified by flash columnchromatography on silica gel (Petroleum Ether/EtOAc=10:1) to affordcompound 2 (420 mg, 36% yield). ¹H NMR (300 MHz, CDCl₃): δ 10.04 (s,1H), 8.36 (d, J=1.8 Hz, 1H), 8.04 (dd, J₁=2.1 Hz, J2=8.4 Hz, 1H), 7.25(m, 1H), 2.91 (m, 1H), 1.59 (s, 9H), 1.36 (d, J=6.9 Hz, 6H)

tert-Butyl 5-(hydroxymethyl)-2-(isobutyryloxy)benzoate (3)

To a solution of compound 2 (400 mg, 1.37 mmol) in THF (20 mL) was addedNaBH₄ (57.3 mg, 1.5 mol) at RT. The reaction mixture was stirred for 1hour and then was quenched by acetone (1 mL). An excess of solvent wasconcentrated and the residue was purified by flash column chromatographyon silica gel (Petroleum Ether/EtOAc=8:1) to afford compound 3 (300 mg,75% yield). ¹H NMR (300 MHz, CDCl₃): δ 7.85 (m, 1H), 7.52 (d, J=8.1 Hz,1H), 7.06 (d, J=8.1 Hz, 1H), 4.73 (s, 2H), 2.88 (m, 1H), 1.57 (s, 9H),1.30 (m, 6H).

5-(Hydroxymethyl)-2-(isobutyryloxy)benzoic acid (4)

A solution of compound 3 (400 mg, 1.38 mmol) in TFA/DCM (v/v, 3 mL/12mL) was stirred at RT overnight. The mixture was poured into water andthe aqueous solution was adjusted to pH=3-4. The two phases wereseparated and the organic phase was dried over anhydrous MgSO₄ andconcentrated to afford compound 4 (202 mg, 62% yield), which was used inthe next step without further purification.

4-(Hydroxymethyl)-2-(prop-2-ynylcarbamoyl)phenyl isobutyrate (5)

To a solution of compound 4 (202 mg, 0.85 mmol) in DCM (20 mL) wereadded DIPEA (219.3 mg, 1.7 mmol), HATU (969 mg, 2.55 mmol) andprop-2-yn-1-amine (93.5 mg, 1.7 mmol) at 0° C. The reaction mixture wasstirred for 30 min. An excess of solvent was concentrated and theresidue was purified by flash column chromatography on silica gel(Petroleum Ether/EtOAc=5:1) to afford compound 5 (130 mg, 60% yield); ¹HNMR (300 MHz, CDCl₃): δ 7.83 (m, 1H), 7.52 (m, 1H), 7.10 (m, 1H), 4.65(s, 2H), 4.24 (m, 2H), 2.87 (m, 1H), 2.30 (m, 1H), 1.26 (m, 6H).

4-(Bromomethyl)-2-(prop-2-ynylcarbamoyl)phenyl isobutyrate (6)

To a solution of compound 5 (130 mg, 0.5 mmol) in DCM (15 mL) were addedPPh3 (170.3 mg, 0.65 mmol) and NBS (105.6 mg, 0.6 mmol) at 0° C. Thereaction mixture was stirred for 30 min and the solvent wasconcentrated. The residue was purified by flash column chromatography onsilica gel (Petroleum Ether/EtOAc=10:1) to afford compound 6 (50 mg, 32%yield). ¹H NMR (300 MHz, CDCl₃): δ 7.88 (m, 1H), 7.37 (m, 1H), 7.11 (m,1H), 4.51 (s, 2H), 4.23 (m, 2H), 2.87 (m, 1H), 2.31 (m, 1H), 1.37 (m,6H).

4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(4-(isobutyryloxy)-3-(prop-2-yn-1-ylcarbamoyl)benzyl)morpholin-4-iumbromide (8)

To a solution of compound 6 (360 mg, 1.1 mmol) in MeCN (4 mL) was added(S)-4-methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide(720 mg, 1.0 mmol). The reaction mixture was stirred at 45° C.overnight. The solvent was concentrated and the residue was purified byflash column chromatography on silica gel (EtOAc/MeOH=50:1) to afforddesired product 7, which was then transformed into the correspondingmesylate (150 mg, 15% yield) by treatment with ion exchange resin; ¹HNMR (400 MHz, CDCl₃): δ 9.61 (m, 1H), 7.90 (m, 1H), 7.64 (m, 2H),7.34-7.10 (m, 12H), 6.92 (m, 1H), 6.68 (m, 1H), 5.03 (m, 2H), 4.86 (m,1H), 4.58-4.32 (m, 4H), 4.28-4.10 (m, 5H), 3.96 (m, 4H), 3.47-3.31 (m,2H), 3.18 (m, 1H), 3.06-2.87 (m, 2H), 2.83 (s, 3H), 2.76 (m, 2H), 2.26(m, 1H), 2.23-2.04 (m, 3H), 1.66-1.58 (m, 2H), 1.42 (m, 4H), 1.38-1.30(m, 6H), 1.27 (m, 4H), 0.90-0.84 (m, 12H).

The pegylated carfilzomib compound Example 4 was prepared from compound8 and PEG_(5K)N₃ following general pegylation procedure A.

Example 5:4-(4-Acetoxy-3-((1-PEG_(5K)-1H-1,2,3-triazol-4-yl)methoxy)benzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholin-4-iummethanesulfonate (7)

4-Hydroxy-3-(prop-2-ynyloxy)benzaldehyde (1)

To a mixture of NaH in DMSO (300 mL) was added 3,4-dihydroxybenzaldehyde(30 g, 217.39 mmol) in DMSO (50 mL) at 20° C. The mixture was stirredfor 30 min and 3-bromoprop-1-yne (25.87 g, 217.39 mmol) was added. Thereaction mixture was stirred at RT for one hour. The mixture was pouredinto ice water (800 mL) and the resulting solution was adjusted to pH=2.The mixture was extracted with EtOAc (500 mL×3), dried over anhydrousMgSO₄, and concentrated. The residue was crystallized repeatedly fromDCM/Petroleum Ether (30 mL/500 mL) to afford compound 1 (30 g, 78%yield); ¹H NMR (CDCl₃, 300 MHz,): δ 9.87 (s, 1H), 7.54 (d, J=1.2 Hz,1H), 7.49 (dd, J, =1.5 Hz, J₂=8.1 Hz, 1H), 7.09 (d, J=8.1 Hz, 1H), 4.82(m, 2H), 2.62 (m, 1H).

4-Formyl-2-(prop-2-ynyloxy)phenyl acetate (2)

To a solution of compound 1 (10.00 g, 56.82 mmol) in DCM (150 mL) wasadded Et₃N (11.48 g, 113.64 mmol) followed by acetyl chloride (5.35 g,68.18 mmol) at 0° C. The reaction mixture was stirred at RT for 2 hours.The mixture was washed with saturated 2 N aqueous HCl (100 mL) and water(50 mL), dried over anhydrous MgSO₄, and concentrated to afford compound2 (12 g, 97% yield), which was used in the next step without furtherpurification; ¹H NMR (CDCl₃, 400 MHz): δ 9.96 (s, 1H), 7.63 (d, J=1.6Hz, 1H), 7.54 (dd, J₁=1.6 Hz, J₂=8.0 Hz, 1H), 7.25 (d, J=8.0 Hz, 1H),4.79 (d, J=2.4 Hz, 2H), 2.57 (t, J=2.4 Hz, 1H), 2.35 (s, 3H).

4-(Hydroxymethyl)-2-(prop-2-ynyloxy)phenyl acetate (3)

To a solution of compound 2 (12.00 g, 55.05 mmol) in DCM/MeOH (150 mL/15mL) was added NaBH₄ (3.06 g, 82.57 mmol) in small portions at 0° C. Thereaction mixture was stirred at RT for 30 min. The mixture was quenchedby acetone (5 mL), and concentrated. The residue was purified by flashcolumn chromatography on silica gel (Petroleum Ether/EtOAc=2:1) toafford compound 3 (10.32 g, 85% yield); ¹H NMR (CDCl₃, 400 MHz): δ 7.16(d, J=1.6 Hz, 1H), 7.04 (d, J=8.0 Hz, 1H), 6.98 (dd, J₁=1.6 Hz, J2=8.0Hz, 1H), 4.72 (d, J=2.4 Hz, 2H), 4.69 (s, 2H), 2.53 (t, J=2.4 Hz, 1H),2.32 (s, 3H).

4-(Bromomethyl)-2-(prop-2-ynyloxy)phenyl acetate (4)

To a solution of compound 3 (11.50 g, 52.27 mmol) in DCM (150 mL) wereadded PPh₃ (20.50 g, 78.41 mmol) and NBS (11.04 g, 62.73 mmol) at 0° C.The reaction mixture was stirred at RT for 0.5 hour. An excess ofsolvent was concentrated and the residue was purified by flash columnchromatography on silica gel (Petroleum Ether/EtOAc=20:1) to affordcompound 4 (7.82 g, 53% yield); ¹H NMR (CDCl₃, 400 MHz): δ 7.14 (m, 1H),7.02 (m, 2H), 4.73 (d, J=2.4 Hz, 2H), 4.48 (s, 2H), 2.55 (t, J=2.4 Hz,1H), 2.32 (s, 3H).

4-(4-Acetoxy-3-(prop-2-yn-1-yloxy)benzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholin-4-iummethanesulfonate (6)

To a solution of compound 4 (5.85 g, 20.67 mmol) in MeCN (50 mL) wasadded(S)-4-methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide(4.96 g, 6.89 mmol). The reaction mixture was stirred at 45° C. for 2days. An excess of solvent was concentrated and the residue was purifiedby flash column chromatography on silica gel (EtOAc/MeOH=100:6) toafford desired compound 5, which was transformed into the correspondingmesylate (3.2 g, 50% yield) by treatment with ion exchange resin; ¹H NMR(CDCl₃, 400 MHz): δ9.66 (m, 1H), 7.83 (m, 1H), 7.36 (m, 1H), 7.26-7.14(m, 13H), 6.72 (m, 1H), 5.18 (m, 1H), 4.90 (m, 2H), 4.77 (m, 2H),4.53-4.36 (m, 4H), 4.26 (m, 3H), 4.08 (m, 1H), 3.92 (m, 2H), 3.74 (m,1H), 3.46 (m, 1H), 3.35 (m, 1H), 3.13 (m, 1H), 3.04 (m, 2H), 2.81 (s,3H), 2.75 (m, 2H), 2.62 (m, 1H), 2.33 (m, 3H), 2.20 (m, 1H), 2.12 (m,1H), 1.70-1.53 (m, 3H), 1.50-1.33 (m, 5H), 1.25 (m, 2H), 0.90-0.81 (m,12H).

The pegylated carfilzomib compound Example 5 was prepared from compound6 and PEG_(5K)N₃ following general pegylation procedure A.

Example 6:4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(3-((1-PEG_(5K)-1H-1,2,3-triazol-4-yl)methoxy)-4-(propionyloxy)benzyl)morpholin-4-iummethanesulfonate (6)

4-Formyl-2-(prop-2-yn-1-yloxy)phenyl propionate (1)

To a solution of 4-hydroxy-3-(prop-2-ynyloxy)benzaldehyde (2.64 g, 15mmol) in DCM (30 mL) were added TEA (3 g, 30 mmol) and propionylchloride (1.67 g, 18 mmol) at 0° C. The reaction mixture was stirred atRT for one hour. This mixture was quenched with water (50 mL) and theDCM phase was collected, dried over anhydrous MgSO₄, and concentrated.The residue was purified by flash column chromatography on silica gel(Petroleum Ether/EtOAc=10:1) to afford compound 1 (2.4 g, 85% yield); ¹HNMR (300 MHz, CDCl₃): δ 9.97 (s, 1H), 7.64 (d, J=1.5 Hz, 1H), 7.55 (d,J1=1.5 Hz, J2=7.8 Hz, 1H), 7.26 (d, J=8.1 Hz, 1H), 4.79 (d, J=2.1 Hz,1H), 2.68 (q, J=7.5 Hz, 2H), 2.58 (t, J=2.4 Hz, 1H), 1.31 (t, J=7.5 Hz,1H).

4-(Hydroxymethyl)-2-(prop-2-ynyloxy)phenyl propionate (2)

To a solution of compound 1 (2.32 g, 0.01 mol) in THF (30 mL) was addedNaBH₄ (570 mg, 0.015 mol) at 0° C. in small portions. The reactionmixture was stirred at room temperature for 2 hours and then wasquenched by saturated NH₄Cl (15 mL). The organic phase was collected andthe aqueous phase was extracted by DCM (20 mL×3). The organic phaseswere combined, dried over anhydrous MgSO₄, and concentrated. The residuewas purified by flash column chromatography on silica gel (PetroleumEther/EtOAc=2:1) to afford compound 2 (1.7 g, 73% yield); ¹H NMR (400MHz, CDCl₃): δ 7.1˜26.95 (m, 3H), 4.68 (m, 2H), 4.62 (m, 2H), 2.63 (m,2H), 2.53 (m, 1H), 1.27 (m, 3H).

4-(Bromomethyl)-2-(prop-2-ynyloxy)phenyl propionate (3)

To a solution of compound 2 (1.7 g, 7.26 mmol) in DCM (30 mL) were addedPPh₃ (2.28 g, 8.7 mmol) and DIPEA (1.12 g, 8.7 mmol) sequentially. Themixture was cooled to 0° C. and NBS (1.4 g, 7.78 mmol) was added insmall portions. The reaction mixture was stirred at the same temperaturefor 20 min. An excess of solvent was concentrated and the residue waspurified by flash column chromatography on silica gel (PetroleumEther/EtOAc=10:1) to afford compound 3 (400 mg, 19% yield).

4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(3-(prop-2-yn-1-yloxy)-4-(propionyloxy)benzyl)morpholin-4-iummethanesulfonate (5)

To a solution of compound 3 (400 mg, 1.34 mmol) in MeCN (5 mL) was addedcompound(S)-4-methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide(484.8 mg, 0.67 mmol). The reaction mixture was heated at 45° C.overnight. An excess of solvent was concentrated and the residue waspurified by flash column chromatography on silica gel to afford thedesired compound 4 (380 mg, 48% yield), which was transformed into thecorresponding mesylate (370 mg, quantitative) by treatment with ionexchange resin; ¹H NMR (400 MHz, CDCl₃): δ 9.63 (m, 1H), 7.82 (m, 1H),7.35˜7.09 (m, 13H), 6.91 (m, 1H), 6.52 (m, 1H), 5.13 (m, 1H), 5.02˜4.82(m, 5H), 4.72 (m, 2H), 4.50˜3.83 (m, 11H), 3.52˜3.31 (m, 2H), 3.18˜2.58(m, 11H), 1.68˜1.18 (m, 9H), 0.88 (m, 12H).

The pegylayted carfilzomib compound Example 6 was prepared from compound5 and PEG_(5K)N₃ following general pegylation procedure A.

Example 7:4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(4-(isobutyryloxy)-3-((1-PEG_(5K)-1H-1,2,3-triazol-4-yl)methoxy)benzyl)morpholin-4-iummethanesulfonate (6)

4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(4-(isobutyryloxy)-3-(prop-2-yn-1-yloxy)benzyl)morpholin-4-iummethanesulfonate (5)

To a solution of compound 3 (0.5 g, 1.6 mmol) in MeCN (9 mL) was added(5)-4-methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide(864 mg, 1.2 mmol). The reaction mixture was stirred at 45° C. for 20hours. The solvent was concentrated and the residue was purified byflash column chromatography on silica gel (EtOAc/MeOH=100:6) to afforddesired compound 4, which was then transformed into the correspondingmesylate (500 mg, 44% yield) by treatment with ion exchange resin; ¹HNMR (400 MHz, CDCl₃): δ 9.67 (m, 1H), 7.82 (m, 1H), 7.27 (m, 16H), 6.85(m, 1H), 6.47 (m, 1H), 5.13 (m, 1H), 5.02 (m, 1H), 4.85 (m, 1H), 4.70(m, 2H), 4.45 (m, 2H), 4.37 (m, 2H), 4.23 (m, 4H), 3.92 (m, 2H), 3.84(m, 1H), 3.46 (m, 1H), 3.35 (m, 1H), 3.15 (m, 1H), 3.03 (m, 1H), 2.92(m, 1H), 2.80 (s, 3H), 2.73 (m, 2H), 2.58 (m, 1H), 2.20 (m, 1H), 2.12(m, 1H), 1.70 (m, 1H), 1.62 (m, 3H), 1.43 (m, 4H), 1.28 (m, 6H), 1.21(m, 3H), 0.85 (m, 12H).

Example 7 was prepared by methods analogous to those described inExamples 3 and 5, wherein the intermediates were made in similarfashion, and compound 5 and PEG_(5K)N₃ were reacted following generalpegylation procedure A.

Example 8:4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(4-(butyryloxy)-3-((1-PEG_(5K)-1H-1,2,3-triazol-4-yl)methoxy)benzyl)morpholin-4-iummethanesulfonate (6)

4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(4-(butyryloxy)-3-(prop-2-yn-1-yloxy)benzyl)morpholin-4-iummethanesulfonate (5)

To a solution of compound 3 (0.7 g, 2.25 mmol) in MeCN (8 mL) was added(S)-4-methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide(0.8 g, 1.125 mmol). The reaction mixture was stirred at 45° C.overnight. An excess of solvent was concentrated and the residue waspurified by flash column chromatography on silica gel (EtOAc/MeOH=100/3)to afford the desired compound 4 (500 mg, 23.3% yield), which wastransformed into the corresponding mesylate (460 mg, 92% yield) bytreatment with ion exchange resin; ¹H NMR (400 MHz, CDCl₃): δ 9.73 (m,1H), 7.76 (m, 1H), 7.33˜7.10 (m, 13H), 6.91 (m, 1H), 6.52 (m, 1H), 5.18(m, 1H), 5.08˜4.85 (m, 2H), 4.72 (m, 2H), 4.50˜3.78 (m, 11H), 3.52˜3.31(m, 2H), 3.18˜2.58 (m, 11H), 2.18 (m, 2H), 1.68˜1.24 (m, 12H), 0.84 (m,12H).

Example 8 was prepared by methods analogous to those described inExample 3, wherein the intermediates were made in similar fashion (usingpropanoyl chloride to generate the correlary to intermediate 1 shown ineg 3), and compound 5 and PEG_(5K)N₃ were reacted following generalpegylation procedure A.

Example 9:4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(4-(hexanoyloxy)-3-((1-PEG_(5K)-1H-1,2,3-triazol-4-yl)methoxy)benzyl)morpholin-4-iummethanesulfonate (6)

4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(4-(hexanoyloxy)-3-(prop-2-yn-1-yloxy)benzyl)morpholin-4-iummethanesulfonate (5)

To a solution of compound 3 (1.41 g, 4.16 mmol) in MeCN (25 mL) wasadded(S)-4-methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide(1.0 g, 1.39 mmol). The reaction mixture was heated at 40˜45° C.overnight. An excess of solvent was concentrated and the residue waspurified by flash column chromatography on silica gel (EtOAc/MeOH=20:1)to afford the desired compound 4, which was transformed into themesylate salt (570 mg, 41% yield) by treatment with ion exchange resin;¹H NMR (400 MHz, CDCl₃): δ 9.72 (m, 1H), 7.82 (m, 1H), 7.34˜7.15 (m,12H), 7.10 (m, 1H), 6.82 (m, 1H), 6.43 (m, 1H), 5.15 (m, 1H), 4.98 (m,1H), 4.76 (m, 2H), 4.46 (m, 2H), 4.38 (m, 2H), 4.25 (m, 3H), 4.12 (m,1H), 4.01 (m, 2H), 3.85 (m, 2H), 3.47 (m, 1H), 3.36 (m, 1H), 3.15 (m,1H), 3.01 (m, 2H), 2.80 (s, 3H), 2.72 (m, 2H), 2.60 (m, 2H), 2.51 (m,1H), 2.35˜2.14 (m, 4H), 1.76 (m, 2H), 1.55 (m, 2H), 1.48 (m, 4H), 1.37(m, 6H), 1.23 (m, 3H), 0.86 (m, 12H).

Example 9 was prepared by methods analogous to those described inExample 3, wherein the intermediates were made in similar fashion (usingpentanoyl chloride to generate the correlary to intermediate 1 shown ineg 3), and compound 5 and PEG_(5K)N₃ were reacted following generalpegylation procedure A.

Example 10:4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(3-((1-PEG_(5K)-1H-1,2,3-triazol-4-yl)methoxy)-4-(octanoyloxy)benzyl)morpholin-4-iummethanesulfonate (6)

Example 10 was prepared by methods analogous to those described inExample 3, wherein the intermediates were made in similar fashion (usingheptanoyl chloride and trimethylamine to generate the correlary tointermediate 1 aldehyde shown in eg. 3), and compound 5 and PEG_(5K)N₃were reacted following general pegylation procedure A.

4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(4-(octanoyloxy)-3-(prop-2-yn-1-yloxy)benzyl)morpholin-4-iummethanesulfonate (5)

To a solution of compound 3 (1.53 g, 4.17 mmol) in MeCN (25 mL) wasadded(S)-4-methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide(1.00 g, 1.39 mmol). The reaction mixture was stirred at 40˜45° C.overnight. The solvent was concentrated and the residue was purified byflash column chromatography on silica gel (EtOAc/MeOH=100:5) to affordthe desired compound 4, which was then transformed into correspondingmesylate (620 mg, 44% yield) by treatment with ion exchange resin; ¹HNMR (400 MHz, CDCl₃): δ 9.69 (m, 1H), 7.82 (m, 1H), 7.34˜7.15 (m, 12H),7.10 (m, 1H), 6.88 (m, 1H), 6.51 (m, 1H), 5.15 (m, 1H), 4.98 (m, 1H),4.88 (m, 1H), 4.74 (m, 2H), 4.46 (m, 2H), 4.38 (m, 2H), 4.25 (m, 4H),4.02 (m, 2H), 3.85 (m, 1H), 3.47 (m, 1H), 3.36 (m, 1H), 3.15 (m, 1H),3.01 (m, 2H), 2.83 (s, 3H), 2.72 (m, 2H), 2.60 (m, 3H), 2.35-2.14 (m,3H), 1.76 (m, 2H), 1.55 (m, 2H), 1.48-1.23 (15H), 0.92˜0.78 (12H).

Example 11:4-(4-Acetoxy-3-methyl-5-((1-PEG_(5K)-1H-1,2,3-triazol-4-yl)methoxy)benzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholin-4-iummethanesulfonate (8)

3,4-Dihydroxy-5-methylbenzaldehyde (1)

To a solution of compound 4-hydroxy-3-methoxy-5-methyl-benzaldehyde(2.00 g, 12.04 mmol) in DCM (100 mL) was added BBr₃ (3.02 g, 12.04 mmol)at −78° C. The reaction mixture was stirred at room temperatureovernight. The mixture was quenched with saturated NH₄Cl (100 mL) at−20° C. The two phases were separated and the aqueous solution wasextracted with EtOAc (50 mL). The combined organic phases were driedover anhydrous MgSO₄ and concentrated. The residue was purified by flashcolumn chromatography on silica gel (Petroleum Ether/EtOAc=10:1) toafford compound 1 (1.56 g, 85% yield); ¹H NMR (300 MHz, DMSO-d6): δ9.94(s, 1H), 9.69 (s, 1H), 9.47 (s, 1H), 7.21 (s, 1H), 7.15 (s, 1H), 2.02(s, 3H).

4-Hydroxy-3-methyl-5-(prop-2-ynyloxy)benzaldehyde (2)

To a mixture of NaH (489.12 mg, 20.38 mmol) in DMSO (30 mL) was addedcompound 1 (1.55 g, 10.19 mmol) in DMSO (10 mL) at 0° C. The mixture wasstirred for 30 min and then 3-bromoprop-1-yne (1.21 g, 10.19 mmol) wasadded at the same temperature. The reaction mixture was stirred for 30min and quenched with water (100 mL). The resulting solution wasadjusted to pH=4-5 and extracted with EtOAc (400 mL×3). The combinedEtOAc phases were washed with brine (50 mL), dried over anhydrous MgSO₄and concentrated. The residue was purified by flash columnchromatography on silica gel (Petroleum Ether/EtOAc=10:1) to affordcompound 2 (1.7 g, 88% yield); ¹H NMR (300 MHz, DMSO-d6): δ 9.84 (s,1H), 9.77 (s, 1H), 7.42 (m, 2H), 4.94 (d, J=2.1 Hz, 2H), 3.65 (m, 1H),2.22 (s, 3H).

4-Formyl-2-methyl-6-(prop-2-ynyloxy)phenyl acetate (3)

To a solution of compound 2 (1.60 g, 8.41 mmol) in DCM was addedpyridine (2.00 g, 25.23 mmol) followed by acetyl chloride (1.32 g, 16.82mmol) in droplet at 0° C. The reaction mixture was stirred at RT for 1hour and then water (100 mL) was added. The two phases were separatedand the organic phase was washed with diluted HCl (1 N, 50 mL), driedover anhydrous MgSO₄ and concentrated to afford compound 3 (2.0 g,quantitative), which was used in the next step without furtherpurification; ¹H NMR (300 MHz, DMSO-d6): δ 9.95 (s, 1H), 7.56 (m, 2H),4.96 (m, 2H), 3.67 (m, 1H), 2.36 (m, 3H), 2.23 (s, 3H).

4-(Hydroxymethyl)-2-methyl-6-(prop-2-ynyloxy)phenyl acetate (4)

To a solution of compound 3 (2.00 g, 8.61 mmol) in THF (50 mL) was addedNaBH₄ (325.80 mg, 8.61 mmol) in small portions at 0° C. The reactionmixture was stirred at the same temperature for 1 hour and then quenchedwith water (1 mL). The mixture was diluted with DCM (100 mL), drieddirectly over anhydrous MgSO₄ and concentrated. The residue was purifiedby flash column chromatography on silica gel (Petroleum Ether/EtOAc=5:1)to afford compound 4 (1.5 g, 74% yield); ¹H NMR (300 MHz, DMSO-d6): δ7.00 (s, 1H), 6.86 (s, 1H), 5.26 (m, 1H), 4.78 (d, J=2.4 Hz, 2H), 4.47(m, 2H), 3.61 (m, 1H), 2.31 (s, 3H), 2.11 (s, 3H).

4-(Bromomethyl)-2-methyl-6-(prop-2-ynyloxy)phenyl acetate (5)

To a solution of compound 4 (1.50 g, 6.46 mmol) in DCM (50 mL) was addedPBr₃ (1.75 g, 6.46 mmol) at 0° C. The reaction mixture was stirred for30 min and then quenched with water (50 mL). The two phases wereseparated and the organic phase was dried over anhydrous MgSO₄ andconcentrated. The residue was purified by flash column chromatography onsilica gel (Petroleum Ether/EtOAc=30:1) to afford compound 5 (750 mg,39% yield); ¹H NMR (300 MHz, CDCl3): δ 7.00 (s, 1H), 6.94 (s, 1H), 4.73(d, J=2.4 Hz, 2H), 4.47 (s, 2H), 2.57 (m, 1H), 2.37 (s, 3H), 2.19 (s,3H).

4-(4-Acetoxy-3-methyl-5-(prop-2-yn-1-yloxy)benzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholin-4-iummethanesulfonate (7)

To a solution of compound 5 (351.25 mg, 1.19 mmol) in MeCN (5 mL) wasadded(S)-4-methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide(428.35 mg, 595.00 umol). The reaction mixture was stirred at 40-45° C.overnight. An excess of solvent was concentrated and the residue waspurified by flash column chromatography on silica gel (EtOAc/MeOH=100:1)to afford desired compound 6, which was transformed into thecorresponding mesylate (280 mg, 50% yield) by treatment with ionexchange resin; ¹H NMR (300 MHz, CDCl₃): δ 9.68 (m, 1H), 7.82 (m, 1H),7.26 (m, 11H), 7.00 (m, 2H), 6.60 (m, 1H), 5.17 (m, 1H), 5.08 (m, 1H),4.78 (m, 3H), 4.46 (m, 4H), 4.22 (m, 4H), 4.01 (m, 2H), 3.80 (m, 2H),3.45 (m, 2H), 3.18 (m, 1H), 3.05 (m, 2H), 2.80 (s, 3H), 2.73 (m, 2H),2.60 (m, 1H), 2.43 (m, 3H), 2.20 (m, 3H), 1.58 (m, 2H), 1.46 (m, 6H),1.32 (m, 3H), 0.88 (m, 12H).

Example 11 was prepared from compound 7 and PEG_(5K)N₃ were reactedfollowing general pegylation procedure A.

Example 12:4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(3-(((1-PEG_(5K)-1H-1,2,3-triazol-4-yl)methyl)carbamoyl)-4-(pivaloyloxy)benzyl)morpholin-4-iummethanesulfonate (9)

4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(4-(pivaloyloxy)-3-(prop-2-yn-1-ylcarbamoyl)benzyl)morpholin-4-iummethanesulfonate (8)

To a solution of compound 6 (400 mg, 1.1 mmol) in MeCN (10 mL) was added(S)-4-methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide(720 mg, 0.1 mmol). The reaction mixture was stirred at 45° C.overnight. An excess of solvent was concentrated and the residue wasrepeatedly crystallized from MeCN/Et₂O (v/v, 1/5) to afford desiredcompound 7, which was transformed into the corresponding mesylate (120mg, 11.2% yield) by treatment with ion exchange resin; ¹H NMR (400 MHz,CDCl₃): δ 9.63 (m, 1H), 7.82˜7.55 (m, 4H), 7.33˜7.08 (m, 11H), 6.85 (m,1H), 6.62 (m, 1H), 5.13˜4.82 (m, 2H), 4.50˜3.93 (m, 14H), 3.42˜2.68 (m,11H), 2.5˜1.9 (m, 6H), 1.68˜1.18 (m, 19H), 0.88 (m, 12H).

Example 12 was prepared by methods analogous to those described inExample 4, wherein the intermediates were made in similar fashion (usingt-butanoyl chloride to generate the correlary to intermediate 1 shown ineg 4), and compound 8 and PEG_(5K)N₃ were reacted following generalpegylation procedure A.

Example 13:4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(3-((1-(PEG_(20K)-4-Arm)-1H-1,2,3-triazol-4-yl)methoxy)-4-(pivaloyloxy)benzyl)morpholin-4-iumformate (7)

4-Hydroxy-3-(prop-2-ynyloxy)benzaldehyde (1)

To a mixture of NaH in DMSO (300 mL) was added 3,4-dihydroxybenzaldehyde(30 g, 217.39 mmol) in DMSO (50 mL) at 20° C. The mixture was stirredfor 30 min and 3-bromoprop-1-yne (25.87 g, 217.39 mmol) was added. Thereaction mixture was stirred at RT for one hour and then poured into icewater. The resulting solution was adjusted to pH=2 and then extractedwith EtOAc (500 mL×3). The combined organic phases were dried overanhydrous MgSO₄ and concentrated. The residue was repeatedlycrystallized from DCM/Petroleum Ether (30 mL/500 mL) to afford compound1 (30 g, 78% yield); ¹H NMR (CDCl₃, 300 MHz,): δ 9.89 (s, 1H), 7.54 (d,J=1.2 Hz, 1H), 7.49 (dd, J₁=1.5 Hz, J₂=8.1 Hz, 1H), 7.09 (d, J=8.1 Hz,1H), 4.82 (m, 2H), 2.62 (m, 1H).

4-Formyl-2-(prop-2-ynyloxy)phenylpivalate (2)

To a solution of compound 1 (3.0 g, 17 mmol) in DCM (120 mL) was addedEt₃N (3.45 g, 34 mmol) followed by pivaloyl chloride (2.34 g, 20.4 mmol)at 0° C. The reaction mixture was stirred at RT for 2 hours. The mixturewas washed with saturated NaHCO₃ (20 mL) and water (20 mL), dried overanhydrous MgSO₄, and concentrated. The residue was purified by flashcolumn chromatography on silica gel (Petroleum Ether/EtOAc=50:1) toafford compound 2 (2.10 g, 47% yield) as a white solid; ¹H NMR (CDCl₃,300 MHz): δ 9.99 (s, 1H), 7.61 (d, J=1.8 Hz, 1H), 7.55 (dd, J₁=1.8 Hz,J2=8.1 Hz, 1H), 7.26 (d, J=8.1 Hz, 1H), 4.77 (d, J=2.4 Hz, 2H), 2.58 (t,J=2.4 Hz, 1H), 1.42 (s, 9H).

4-(Hydroxymethyl)-2-(prop-2-ynyloxy)phenylpivalate (3)

To a solution of compound 2 (1.8 g, 6.9 mmol) in DCM/MeOH (100 mL/10 mL)was added NaBH₄ (0.37 g, 10.4 mmol) at 0° C. The reaction mixture wasstirred at RT for 30 min. The mixture was quenched by acetone (3 mL) andthe solvent was concentrated. The residue was purified by flash columnchromatography on silica gel (Petroleum Ether/EtOAc=3:1) to affordcompound 3 (1.50 g, 83% yield); ¹H NMR (CDCl₃, 300 MHz): δ 7.11 (m, 1H),7.00 (m, 2H), 4.68 (m, 4H), 2.53 (m, 1H), 1.41 (s, 9H).

4-(Bromomethyl)-2-(prop-2-ynyloxy)phenylpivalate (4)

To a solution of compound 3 (1.50 g, 5.7 mmol) in DCM (60 mL) were addedPPh₃ (1.80 g, 6.8 mmo) and NBS (1.11 g, 6.3 mmol) at 0° C. The reactionmixture was stirred at RT for 0.5 hour. An excess of solvent wasconcentrated and the residue was purified by flash column chromatographyon silica gel (Petroleum Ether/EtOAc=50:1) to afford compound 4 (1.34 g,81% yield); ¹H NMR (CDCl₃, 300 MHz): δ 7.12 (d, J=1.5 Hz, 1H), 7.03 (m,2H), 4.70 (d, J=2.4 Hz, 2H), 4.51 (d, J=3.9 Hz, 2H), 2.56 (t, J=2.4 Hz,1H), 1.40 (s, 9H).

4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(4-(pivaloyloxy)-3-(prop-2-yn-1-yloxy)benzyl)morpholin-4-iummethanesulfonate (6)

To a solution of compound 4 (2.38 g, 7.3 mmol) in MeCN (30 ml) was added(S)-4-methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide(2.64 g, 3.7 mmol). The reaction mixture was stirred at 45° C.overnight. An excess of solvent was concentrated and the residue waspurified by flash column chromatography on silica gel (EtOAc/MeOH=100:6)to afford desired compound 5, which was transformed into thecorresponding mesylate (1.23 g, 25% yield) by treatment with ionexchange resin; ¹H NMR (CDCl₃, 300 MHz): δ 9.83 (m, 1H), 7.92 (m, 1H),7.50-7.11 (m, 13H), 7.03 (m, 1H), 6.62 (m, 1H), 5.25 (m, 1H), 5.15-4.90(m, 2H), 4.88-4.75 (m, 2H), 4.70-4.20 (m, 7H), 4.20-3.90 (m, 3H),3.70-3.40 (m, 4H), 3.26 (m, 1H), 3.15 (m, 2H), 2.90 (s, 3H), 2.85 (m,2H), 2.40-2.10 (m, 2H), 1.87-1.63 (m, 5H), 1.55 (m, 3H), 1.41 (s, 9H),1.38 (m, 2H), 0.89-1.05 (m, 12H).

Compound Example 13 was prepared from compound 6 and PEG_(20K)(N₃)₄following general pegylation procedure A. Compound Example 13 is alsodesignated as OP-59381 in various of the figures illustrated herein. ¹HNMR (500 MHz, relaxation time=10 sec, DMSO-d₆) δ 8.47 (s, 4H), 8.42 (d,J=8.5 Hz, 4H), 8.29 (d, J=7.5 Hz, 4H), 8.11 (s, 4H), 8.07 (d, J=8 Hz,4H), 7.53 (s, 4H), 7.26-7.29 (m, 4H), 7.11-7.19 (m, 32H), 7.05-7.06 (m,4H), 5.21 (s, 8H), 4.95 (dd, J=12.5 Hz and 39.0 Hz, 8H), 4.52-4.54 (m,12H), 4.28-4.38 (m, 16H), 4.17-4.20 (m, 4H), 4.06 (m, 20H), 3.78 (t,J=5.5 Hz, 8H), 3.61-3.65 (m, 8H), 3.50 (s, 2133H), 3.35-3.37 (m, 8H),3.10 (d, J=5 Hz, 4H), 2.94-2.98 (m, 12H), 2.73-2.78 (m, 4H), 2.50-2.65(m, 8H), 1.90-1.98 (m, 4H), 1.78-1.88 (m, 4H), 1.51-1.68 (m, 8H), 1.39(s, 12H), 1.25-1.38 (m, 16H), 1.18 (s, 36H), 0.833-0.881 (m, 24H),0.782-0.815 (m, 24H); Loading: 86%.

Example 14:4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(4-(isobutyryloxy)-3-((1-PE-4-Arm-1H-1,2,3-triazol-4-yl)methoxy)benzyl)morpholin-4-iummethanesulfonate (14)

4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(4-(isobutyryloxy)-3-(prop-2-yn-1-yloxy)benzyl)morpholin-4-iummethanesulfonate (5)

To a solution of compound 3 (0.5 g, 1.6 mmol) in MeCN (9 mL) was added(S)-4-methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide(864 mg, 1.2 mmol). The reaction mixture was stirred at 45° C. for 20hours. The solvent was concentrated and the residue was purified byflash column chromatography on silica gel (EtOAc/MeOH=100:6) to afforddesired compound 4, which was then transformed into the correspondingmesylate (500 mg, 44% yield) by treatment with ion exchange resin; ¹HNMR (400 MHz, CDCl₃): δ 9.67 (m, 1H), 7.82 (m, 1H), 7.27 (m, 16H), 6.85(m, 1H), 6.47 (m, 1H), 5.13 (m, 1H), 5.02 (m, 1H), 4.85 (m, 1H), 4.70(m, 2H), 4.45 (m, 2H), 4.37 (m, 2H), 4.23 (m, 4H), 3.92 (m, 2H), 3.84(m, 1H), 3.46 (m, 1H), 3.35 (m, 1H), 3.15 (m, 1H), 3.03 (m, 1H), 2.92(m, 1H), 2.80 (s, 3H), 2.73 (m, 2H), 2.58 (m, 1H), 2.20 (m, 1H), 2.12(m, 1H), 1.70 (m, 1H), 1.62 (m, 3H), 1.43 (m, 4H), 1.28 (m, 6H), 1.21(m, 3H), 0.85 (m, 12H).

Example 14 was prepared by methods analogous to those described inExample 3, 5 and 7 wherein the intermediates were made in similarfashion (using isopropanoyl chloride to generate the correlary tointermediate 1 shown in eg 3), and compound 5 and PEG_(20K)N₃ werereacted following general pegylation procedure A.

Example 15:4-(4-Acetoxy-3-(2-(2-(2-((1-PEG_(5K)-1H-1,2,3-triazol-4-yl)methoxy)ethoxy)ethoxy)ethoxy)benzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholin-4-iummethanesulfonate (9)

2-(2-(2-(Prop-2-yn-1-yloxy)ethoxy)ethoxy)ethan-1-ol (1)

To a mixture of NaH (3.47 g, 0.086 mol) in THF (320 mL) was added2,2′-(ethane-1,2-diylbis(oxy))diethanol (20 g, 0.133 mol) at 0° C. Themixture was stirred at the same temperature for 30 min and then3-bromoprop-1-yne (7.93 g, 0.066 mol) was added. The reaction mixturewas kept at the 0° C. for 2 hours and then left to RT overnight. Themixture was quenched with water (4 mL) and the resulting solution wasdried over anhydrous MgSO₄ directly and concentrated. The residue waspurified by flash column chromatography on silica gel (PetroleumEther/EtOAc=1:1) to afford compound 1 (10.12 g, 80% yield); ¹H NMR (400MHz, CDCl₃): δ 4.21 (d, J=2.0 Hz, 2H), 3.75-3.68 (m, 10H), 3.62 (m, 2H),2.44 (m, 1H), 2.23 (s, 1H).

2-(2-(2-(Prop-2-yn-1-yloxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate(2)

To a solution of compound 1 (5 g, 26.6 mmol) in DCM (80 mL) was addedTsCl (7.6 g, 39.89 mmol) at 0° C. followed by pyridine (25 mL). Thereaction mixture was stirred at RT overnight. The DCM solution waswashed with HCl (3 N, 50 mL×4), dried and concentrated to affordcompound 2 (7.89 g, 87% yield), which was used in the next step withoutfurther purification; ¹H NMR (400 MHz, CDCl₃): δ 7.80 (d, J=8.4 Hz, 2H),7.34 (d, J=8.0 Hz, 2H), 4.20 (m, 4H), 3.68 (m, 6H), 3.61 (m, 4H), 2.45(m, 1H), 2.40 (m, 3H).

4-Hydroxy-3-(2-(2-(2-(prop-2-ynyloxy)ethoxy)ethoxy)ethoxy)benzaldehyde(3)

To a mixture of NaH (0.82 g, 20.47 mmol) in DMSO (50 mL) were added asolution of 3,4-dihydroxybenzaldehyde (1.41 g, 10.23 mmol) in DMSO (5mL) and a solution of compound 2 (3.5 g, 10.23 mmol) in DMSO (5 mL) at20° C. sequentially. The reaction mixture was stirred at RT overnight.The mixture was poured into ice water (500 mL) and this aqueous solutionwas adjusted to pH=2 by 2 N HCl. The resulting mixture was extractedwith EtOAc (50 mL×3) and the combined EtOAc phases were dried overanhydrous MgSO₄ and concentrated. The residue was purified by flashcolumn chromatography on silica gel (Petroleum Ether/EtOAc=1:1) toafford compound 3 (579 mg, 18% yield); ¹H NMR (400 MHz, CDCl₃): δ 9.80(s, 1H), 7.46 (m, 2H), 7.03 (d, J=7.6 Hz, 1H), 4.25 (m, 4H), 3.88 (m,2H), 3.70 (m, 8H), 2.45 (m, 1H).

4-Formyl-2-(2-(2-(2-(prop-2-ynyloxy)ethoxy)ethoxy)ethoxy)phenyl acetate(4)

To a solution of compound 3 (479 mg, 1.56 mmol) in THF (20 mL) wereadded TEA (471 mg, 4.67 mmol) and Ac₂O (238 mg, 2.33 mmol) at 0° C. Thereaction mixture was stirred at room temperature for 3 hours. An excessof solvent was concentrated and the residue was dissolved in EtOAc (40mL). The resulting solution was washed with water (50 mL), dried overanhydrous MgSO₄ and concentrated. The residue was purified by flashcolumn chromatography on silica gel (Petroleum Ether/EtOAc=2:1) toafford compound 4 (400 mg, 74% yield); ¹H NMR (400 MHz, CDCl₃): δ 9.94(s, 1H), 7.52 (d, J=1.6 Hz, 1H), 7.49 (dd, J₁=1.6 Hz, J2=8.0 Hz, 1H),7.22 (d, J=8.0 Hz, 1H), 4.23 (m, 2H), 4.20 (m, 2H), 3.86 (m, 2H), 3.72(m, 8H), 2.43 (m, 1H), 2.34 (s, 3H).

4-(Hydroxymethyl)-2-(2-(2-(2-(prop-2-ynyloxy)ethoxy)ethoxy)ethoxy)phenylacetate (5)

To a solution of compound 4 (1.18 g, 3.38 mmol) in THF (50 mL) was addedBH₃/THF solution (3.4 mL, 3.38 mmol) dropwise at 0° C. The reactionmixture was stirred for 30 min and then quenched with MeOH (5 mL). Thereaction solution was concentrated and the residue was purified by flashcolumn chromatography on silica gel (Petroleum Ether/EtOAc=1:1) toafford compound 5 (700 mg, 59% yield); ¹H NMR (300 MHz, CDCl₃): δ 7.09(d, J=1.8 Hz, 1H), 7.03 (d, J=8.1 Hz, 1H), 6.94 (dd, J₁=1.8 Hz, J2=8.1Hz, 1H), 4.68 (s, 2H), 4.22 (m, 4H), 3.85 (m, 2H), 3.74 (m, 8H), 2.46(m, 1H), 2.33 (s, 3H).

4-(Bromomethyl)-2-(2-(2-(2-(prop-2-ynyloxy)ethoxy)ethoxy)ethoxy)phenylacetate) (6)

To a solution of compound 5 (680 mg, 1.93 mmol) in DCM (40 mL) was addedPPh₃ (607 mg, 2.32 mmol) followed by NBS (374 mg, 2.13 mmol) in smallportions at 0° C. An excess of solvent was concentrated and the residuewas purified by flash column chromatography on silica gel (PetroleumEther/EtOAc=3:1) to afford compound 6 (430 mg, 54% yield); ¹H NMR (400MHz, CDCl₃): δ 7.03 (s, 1H), 6.99 (m, 2H), 4.46 (s, 2H), 4.20-4.16 (m,4H), 3.83 (m, 2H), 3.68 (m, 8H), 2.43 (m, 1H), 2.29 (s, 3H). Compound 6was converted to compound 8 using analogous methods as described herein.

4-(4-Acetoxy-3-(2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxyl)ethoxy)benzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholin-4-iummethanesulfonate (8)

To a solution of compound 6 (430 mg, 1.04 mmol) in MeCN (5 mL) was addedcompound(S)-4-methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide(743 mg, 1.04 mmol). The reaction mixture was stirred at RT overnight.An excess of solvent was concentrated and the residue was purified byflash column chromatography on silica gel (EtOAc/MeOH=10:1) to afforddesired product 7 (160 mg, 14% yield), which was then transformed to thecorresponding mesylate salt (135 mg, 85% yield) by treatment with ionexchange resin; ¹H NMR (400 MHz, CDCl₃): δ 9.68 (m, 1H), 7.73 (m, 1H),7.29-7.05 (m, 13H), 6.82 (m, 1H), 6.40 (m, 1H), 5.15 (m, 1H), 5.08 (m,1H), 5.02 (m, 1H), 4.82 (m, 2H), 4.51 (m, 2H), 4.38 (m, 3H), 4.20 (m,4H), 4.15 (m, 2H), 4.03 (m, 2H), 3.84 (m, 1H), 3.76 (m, 2H), 3.65 (m,9H), 3.50 (m, 1H), 3.38 (m, 1H), 3.18 (m, 1H), 3.02 (m, 2H), 2.86 (m,1H), 2.80 (s, 3H), 2.64 (m, 2H), 2.46 (m, 1H), 2.32 (m, 3H), 2.30-2.05(m, 3H), 1.60 (m, 2H), 1.52 (m, 6H), 1.24 (m, 2H), 0.84 (12H).

Example 15 was prepared from compound 8 and PEG_(5K)N₃ following generalpegylation procedure A.

Example 16:4-(4-Acetoxy-3-(1-(PEG_(5K)-imino)ethyl)benzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholin-4-iummethanesulfonate (5)

2-Acetyl-4-methylphenyl acetate (1)

To a solution of 1-(2-hydroxy-5-methylphenyl)ethanone (1.5 g, 0.01 mol)in DCM (15 mL) were added TEA (1.5 g, 0.015 mol) and acetyl chloride(0.94 g, 0.012 mol) at 0° C. The reaction mixture was stirred at roomtemperature overnight. This mixture was quenched with water (20 mL). TheDCM phase was collected, washed with brine (20 mL), dried over anhydrousMgSO₄, and concentrated. The residue was purified by flash columnchromatography on silica gel (Petroleum Ether/EtOAc=3:1) to affordcompound 1 (0.9 g, 47% yield); ¹H NMR (300 MHz, CDCl₃): δ 7.64 (m, 1H),7.37 (m, 1H), 7.02 (d, J=8.1 Hz, 1H), 2.57 (s, 3H), 2.42 (s, 3H), 2.37(s, 3H).

2-Acetyl-4-(bromomethyl)phenyl acetate (2)

To a solution of compound 1 (0.5 g, 2.6 mmol) in CCl₄ (20 mL) were addedNBS (573 mg, 3.25 mmol) and AIBN (42.6 mg, 0.26 mmol). The reactionmixture was heated under reflux overnight. The mixture was cooled to RTand filtered. The filtrate was concentrated and the residue was purifiedby flash column chromatography on silica gel (PetroleumEther/EtOAc=10:1) to afford compound 2 (160 mg, 23% yield); ¹H NMR (300MHz, DMSO-d6): δ 8.02 (m, 1H), 7.73 (d, J=8.1 Hz, 1H), 7.25 (d, J=8.4Hz, 1H), 4.81 (s, 2H), 2.53 (s, 3H), 2.32 (s, 3H).

4-(4-Acetoxy-3-acetylbenzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholin-4-iummethanesulfonate (4)

To a solution of compound 2 (1.03 g, 3.7 mmol) in MeCN (10 mL) was addedcompound(S)-4-methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide(884.7 mg, 1.23 mmol). The reaction mixture was heated at 45° C.overnight. An excess of solvent was concentrated and the residue waspurified by flash column chromatography on silica gel (EtOAc/MeOH=100:3)to afford the desired compound 3, which was transformed into thecorresponding mesylate (260 mg, 21% yield) by treatment with ionexchange resin; ¹H NMR (300 MHz, CDCl₃): δ 9.62 (m, 1H), 8.06 (m, 1H),7.88˜7.71 (m, 2H), 7.33˜7.11 (m, 11H), 6.95 (m, 1H), 6.66 (m, 1H),5.33˜4.91 (m, 2H), 4.55˜3.90 (m, 11H), 3.58˜2.91 (m, 4H), 2.85 (s, 3H),2.74 (m, 2H), 2.61 (s, 3H), 2.40 (s, 3H), 2.31˜1.94 (m, 7H), 1.72˜1.18(m, 8H), 0.88 (m, 12H).

Example 16 was prepared from compound 4 and PEG_(5K)ONH₃ ⁺.MsO⁻following general pegylation procedure B.

Example 17:4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-β-(1-(PEG_(5K)-imino)ethyl)-4-(pivaloyloxy)benzyl)morpholin-4-iummethanesulfonate (5)

4-(3-Acetyl-4-(pivaloyloxy)benzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholin-4-iummethanesulfonate (4)

To a solution of compound 2 (1.03 g, 3.2 mmol) in MeCN (10 mL) was added(S)-4-methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide(766 mg, 1.06 mmol). The reaction mixture was stirred at 45° C.overnight. An excess of solvent was concentrated and the residue wasrepeatedly crystallized from EtOAc/Et₂O (5/1, v/v) to afford desiredcompound 3, which was transformed into the corresponding mesylate (300mg, 32% yield) by treatment with ion exchange resin; ¹H NMR (300 MHz,CDCl₃): δ 9.68 (m, 1H), 8.06 (m, 1H), 7.75 (m, 1H), 7.40-7.15 (m, 12H),6.92 (m, 1H), 6.65 (m, 1H), 5.28-4.96 (m, 2H), 4.55-4.42 (m, 4H),4.38-4.18 (m, 4H), 4.07-3.90 (m, 3H), 3.60-3.30 (m, 2H), 3.17 (m, 2H),3.04 (m, 2H), 2.85 (s, 3H), 2.80 (m, 2H), 2.63 (s, 3H), 2.44 (s, 3H),2.28-2.12 (m, 2H), 2.04 (m, 3H), 1.76 (m, 3H), 1.50-1.40 (m, 6H),1.30-1.18 (m, 4H), 0.92-0.84 (m, 12H).

Example 17 was prepared by methods analogous to those described inExample 16, wherein the intermediates were made in similar fashion(using t-butanoyl chloride to generate the correlary to intermediate 1shown in eg 16), and compound 4 and PEG_(5K)ONH₃ ⁺.MsO⁻ followinggeneral pegylation procedure B.

Example 18:4-(3-Acetoxy-4-((PEG_(5K)-imino)methyl)benzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholin-4-iummethanesulfonate (8)

2-Hydroxy-5-(hydroxymethyl)benzaldehyde (1)

To an aqueous solution of formaldehyde (37%, 17 mL) were added2-hydroxybenzaldehyde (10.3 g, 84.4 mmol) and concentrated HCl (42 mL).The reaction mixture was heated under reflux overnight. The mixture wascooled to RT and then extracted with EtOAc (200 mL). The organic phasewas dried over anhydrous sodium sulfate and concentrated. The residuewas purified by flash column chromatography on silica gel (PetroleumEther/EtOAc=3:1) to afford compound 1 (1.97 g, 15% yield); ¹H NMR(DMSO-d₆, 300 MHz): δ 10.61 (s, 1H), 10.26 (s, 1H), 7.60 (d, J=2.1 Hz,1H), 7.46 (dd, J=2.4, 8.7 Hz, 1H), 6.96 (d, J=8.4 Hz, 1H), 5.18 (m, 1H),4.42 (d, J=3.3 Hz, 2H).

5-(((tert-Butyldimethylsilyl)oxy)methyl)-2-hydroxybenzaldehyde (2)

To a solution of compound 1 (2.01 g, 13.2 mmol) in DCM (60 mL) was addedimidazole (1.43 g, 21 mmol). The solution was cooled to 0° C. andtert-butylchloro dimethylsilane (2.57 g, 17.1 mmol) was added. Thereaction mixture was stirred at RT for 3 h and then poured into water(50 mL). The two phases were separated and the organic phase was driedover anhydrous sodium sulfate and concentrated. The residue was purifiedby flash column chromatography on silica gel (PetroleumEther/EtOAc=50:1) to afford compound 2 (3.2 g, 91% yield); ¹H NMR(CDCl₃, 400 MHz): δ 10.85 (br, s, 1H), 9.78 (s, 1H), 7.41 (d, J=2.0 Hz,1H), 7.35 (dd, J=2.0, 8.4 Hz, 1H), 6.85 (d, J=8.4 Hz, 1H), 4.59 (s, 2H),0.82 (s, 9H), 0.00 (s, 6H).

4-((tert-Butyldimethylsilyloxy)methyl)-2-formylphenyl acetate (3)

To a solution of compound 2 (25 g, 94 mmol) in DCM (500 mL) was addedTEA (19.0 g, 188 mmol). The mixture was cooled to 0° C. and acetylchloride (11.1 g, 141 mmol) was added. The reaction mixture was stirredat room temperature for 2 h. The mixture was washed with water (500 mL).The organic phase was dried over anhydrous sodium sulfate andconcentrated. The residue was purified by flash column chromatography onsilica gel (Petroleum Ether/EtOAc=100:1) to afford compound 3 (19.7 g,68% yield); ¹H NMR (CDCl₃, 400 MHz): δ 9.98 (s, 1H), 7.70 (d, J=2.0 Hz,1H), 7.49 (dd, J=2.4, 8.4 Hz, 1H), 7.03 (d, J=2.4 Hz, 1H), 4.66 (s, 2H),2.28 (s, 3H), 0.83 (s, 9H), 0.00 (s, 6H).

2-Formyl-4-(hydroxymethyl)phenyl acetate (4)

Compound 3 (3.6 g, 11.7 mmol) was dissolved in AcOH/THF/H₂O (50 mL/25mL/25 mL). The reaction mixture was stirred at 30° C. for 3 h. An excessof THF was removed and the resulting solution was adjusted to pH=7-8 andthen extracted with EtOAc (50 mL×3). The combined organic phases weredried over anhydrous sodium sulfate and concentrated. The residue waspurified by flash column chromatography on silica gel (PetroleumEther/EtOAc=3:1) to afford compound 4 (2.04 g, 90% yield); ¹H NMR(DMSO-d₆, 400 MHz): δ 10.08 (s, 1H), 7.85 (d, J=2.0 Hz, 1H), 7.67 (dd,J=2.4, 8.4 Hz, 1H), 7.26 (d, J=8.4 Hz, 1H), 4.57 (s, 2H), 2.35 (s, 3H).

4-(Bromomethyl)-2-formylphenyl acetate (5a)

To a solution of compound 4 (2.03 g, 10.3 mmol) in DCM (80 mL) was addedPBr₃ (2.79 g, 10.3 mmol) at 0° C. The reaction mixture was stirred at RTfor 4 h. The reaction was quenched by addition of water (20 mL) and theresulting mixture was adjusted to pH=7 with saturated aqueous NaHCO₃.The organic phase was separated, dried over anhydrous sodium sulfate andconcentrated. The residue was purified by flash column chromatography onsilica gel (Petroleum Ether/EtOAc=3:1) to afford compound 5a (300 mg,11% yield); ¹H NMR (CDCl₃, 300 MHz): δ 10.12 (s, 1H), 7.92 (d, J=2.1 Hz,1H), 7.68 (dd, J=2.4, 8.4 Hz, 1H), 7.22 (d, J=8.1 Hz, 1H), 4.54 (s, 2H),2.42 (s, 3H).

4-(Iodomethyl)-2-formylphenyl acetate (5b)

To a solution of compound 4 (5.0 g, 27.55 mmol) in DCM (300 mL) wasadded SOCl₂ (6.13 g, 51.55 mmol) at 0° C. The reaction mixture washeated under reflux overnight. The mixture was concentrated and theresidue was purified by flash column chromatography on silica gel(Petroleum Ether/EtOAc=10:1) to afford the corresponding benzyl chloride(2.4 g, 44% yield); ¹H NMR (CDCl₃, 300 MHz): δ 10.12 (s, 1H), 7.92 (d,J=2.4 Hz, 1H), 7.68 (dd, J=2.4, 8.4 Hz, 1H), 7.22 (d, J=2.4 Hz, 1H),4.64 (s, 2H), 2.42 (s, 3H).

To a solution of benzyl chloride (2.4 g, 11.29 mmol) in acetone (160 mL)was added NaI (16.94 g, 112.94 mmol). The reaction mixture was stirredat 30° C. overnight. The mixture was concentrated and the residue wasdissolved in DCM (100 mL). The resulting solution was washed withsaturated aqueous Na₂S₂O₃ (50 mL×3) and water (50 mL), dried overanhydrous sodium sulfate and concentrated to afford compound 5b (2.1 g,61% yield), which was used in the next step without furtherpurification. ¹H NMR (CDCl₃, 300 MHz): δ 10.10 (s, 1H), 7.90 (d, J=2.4Hz, 1H), 7.66 (dd, J=2.1, 8.4 Hz, 1H), 7.16 (d, J=2.4 Hz, 1H), 4.49 (s,2H), 2.41 (s, 3H).

4-(4-Acetoxy-3-formylbenzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholin-4-iummethanesulfonate (7)

To a solution of compound 5b (380 mg, 1.48 mmol) in MeCN (5 mL) wasadded(S)-4-methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide(532 mg, 0.74 mmol). The reaction mixture was stirred at 45° C.overnight. The solvent was removed under reduced pressure. The residuewas purified by flash column chromatography on silica gel(DCM/MeOH=10:1) to afford desired compound (6), which was thentransformed into the corresponding mesylate by treatment with ionexchange resin (280 mg, 39% yield); ¹H NMR (CDCl₃, 400 MHz): δ 10.15 (s,1H), 9.53 (br s, 1H), 8.03 (d, J=2.0 Hz, 1H), 7.85 (m, 1H), 7.68 (br s,1H), 7.37 (d, J=8.0 Hz, 1H), 7.26-7.13 (m, 10H), 6.84 (br s, 1H), 6.52(br s, 1H), 5.20 (m, 2H), 4.97 (m, 1H), 4.50-3.96 (m, 7H), 3.46-3.28 (m,2H), 3.16 (m, 1H), 3.06-2.92 (m, 3H), 2.85-2.61 (m, 7H), 2.44 (s, 3H),2.14 (m, 2H), 1.69-1.17 (m, 11H), 0.89-0.83 (m, 12H). Compound 5a couldalso be used for this reaction.

Example 18 was prepared from compound 7 and PEG_(5K)ONH₃ ⁺.MsO⁻following general pegylation procedure A.

Example 19:4-(4-Acetoxy-3-((E)-((2-(PEG_(5K)-amino)-2-oxoethoxy)imino)methyl)benzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholin-4-iummethanesulfonate (8)

2-Hydroxy-5-(hydroxymethyl)benzaldehyde (1)

To an aqueous solution of formaldehyde (37%, 17 mL) were added2-hydroxybenzaldehyde (10.3 g, 84.4 mmol) and concentrated HCl (42 mL).The reaction mixture was heated under reflux overnight. The mixture wascooled to RT and then extracted with EtOAc (200 mL). The organic phasewas dried over anhydrous sodium sulfate and concentrated. The residuewas purified by flash column chromatography on silica gel (PetroleumEther/EtOAc=3:1) to afford compound 1 (1.97 g, 15% yield); ¹H NMR(DMSO-d₆, 300 MHz): δ 10.61 (s, 1H), 10.26 (s, 1H), 7.60 (d, J=2.1 Hz,1H), 7.46 (dd, J=2.4, 8.7 Hz, 1H), 6.96 (d, J=8.4 Hz, 1H), 5.18 (m, 1H),4.42 (d, J=3.3 Hz, 2H).

5-(((tert-Butyldimethylsilyl)oxy)methyl)-2-hydroxybenzaldehyde (2)

To a solution of compound 1 (2.01 g, 13.2 mmol) in DCM (60 mL) was addedimidazole (1.43 g, 21 mmol). The solution was cooled to 0° C. andtert-butylchloro dimethylsilane (2.57 g, 17.1 mmol) was added. Thereaction mixture was stirred at RT for 3 h and then poured into water(50 mL). The two phases were separated and the organic phase was driedover anhydrous sodium sulfate and concentrated. The residue was purifiedby flash column chromatography on silica gel (PetroleumEther/EtOAc=50:1) to afford compound 2 (3.2 g, 91% yield); ¹H NMR(CDCl₃, 400 MHz): δ 10.85 (br, s, 1H), 9.78 (s, 1H), 7.41 (d, J=2.0 Hz,1H), 7.35 (dd, J=2.0, 8.4 Hz, 1H), 6.85 (d, J=8.4 Hz, 1H), 4.59 (s, 2H),0.82 (s, 9H), 0.00 (s, 6H).

4-((tert-Butyldimethylsilyloxy)methyl)-2-formylphenyl acetate (3)

To a solution of compound 2 (25 g, 94 mmol) in DCM (500 mL) was addedTEA (19.0 g, 188 mmol). The mixture was cooled to 0° C. and acetylchloride (11.1 g, 141 mmol) was added. The reaction mixture was stirredat RT for 2 h. The mixture was washed with water (500 mL). The organicphase was dried over anhydrous sodium sulfate and concentrated. Theresidue was purified by flash column chromatography on silica gel(Petroleum Ether/EtOAc=100:1) to afford compound 3 (19.7 g, 68% yield);¹H NMR (CDCl₃, 400 MHz): δ 9.98 (s, 1H), 7.70 (d, J=2.0 Hz, 1H), 7.49(dd, J=2.4, 8.4 Hz, 1H), 7.03 (d, J=2.4 Hz, 1H), 4.66 (s, 2H), 2.28 (s,3H), 0.83 (s, 9H), 0.00 (s, 6H).

2-Formyl-4-(hydroxymethyl)phenyl acetate (4)

Compound 3 (3.6 g, 11.7 mmol) was dissolved in AcOH/THF/H₂O (50 mL/25mL/25 mL). The reaction mixture was stirred at 30° C. for 3 h. An excessof THF was removed and the resulting solution was adjusted to pH=7-8 andthen extracted with EtOAc (50 mL×3). The combined organic phases weredried over anhydrous sodium sulfate and concentrated. The residue waspurified by flash column chromatography on silica gel (PetroleumEther/EtOAc=3:1) to afford compound 4 (2.04 g, 90% yield); ¹H NMR(DMSO-d₆, 400 MHz): δ 10.08 (s, 1H), 7.85 (d, J=2.0 Hz, 1H), 7.67 (dd,J=2.4, 8.4 Hz, 1H), 7.26 (d, J=8.4 Hz, 1H), 4.57 (s, 2H), 2.35 (s, 3H).

4-(Bromomethyl)-2-formylphenyl acetate (5a)

To a solution of compound 4 (2.03 g, 10.3 mmol) in DCM (80 mL) was addedPBr₃ (2.79 g, 10.3 mmol) at 0° C. The reaction mixture was stirred at RTfor 4 h. The reaction was quenched by addition of water (20 mL) and theresulting mixture was adjusted to pH=7 with saturated aqueous NaHCO₃.The organic phase was separated, dried over anhydrous sodium sulfate andconcentrated. The residue was purified by flash column chromatography onsilica gel (Petroleum Ether/EtOAc=3:1) to afford compound 5a (300 mg,11% yield); ¹H NMR (CDCl₃, 300 MHz): δ 10.12 (s, 1H), 7.92 (d, J=2.1 Hz,1H), 7.68 (dd, J=2.4, 8.4 Hz, 1H), 7.22 (d, J=8.1 Hz, 1H), 4.54 (s, 2H),2.42 (s, 3H).

4-(Iodomethyl)-2-formylphenyl acetate (5b)

To a solution of compound 4 (5.0 g, 27.55 mmol) in DCM (300 mL) wasadded SOCl₂ (6.13 g, 51.55 mmol) at 0° C. The reaction mixture washeated under reflux overnight. The mixture was concentrated and theresidue was purified by flash column chromatography on silica gel(Petroleum Ether/EtOAc=10:1) to afford the corresponding benzyl chloride(2.4 g, 44% yield); ¹H NMR (CDCl₃, 300 MHz): δ 10.12 (s, 1H), 7.92 (d,J=2.4 Hz, 1H), 7.68 (dd, J=2.4, 8.4 Hz, 1H), 7.22 (d, J=2.4 Hz, 1H),4.64 (s, 2H), 2.42 (s, 3H).

To a solution of benzyl chloride (2.4 g, 11.29 mmol) in acetone (160 mL)was added NaI (16.94 g, 112.94 mmol). The reaction mixture was stirredat 30° C. overnight. The mixture was concentrated and the residue wasdissolved in DCM (100 mL). The resulting solution was washed withsaturated aqueous Na₂S₂O₃ (50 mL×3) and water (50 mL), dried overanhydrous sodium sulfate and concentrated to afford compound 5b (2.1 g,61% yield), which was used in the next step without furtherpurification. ¹H NMR (CDCl₃, 300 MHz): δ 10.10 (s, 1H), 7.90 (d, J=2.4Hz, 1H), 7.66 (dd, J=2.1, 8.4 Hz, 1H), 7.16 (d, J=2.4 Hz, 1H), 4.49 (s,2H), 2.41 (s, 3H).

4-(4-Acetoxy-3-formylbenzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholin-4-iummethanesulfonate (7)

To a solution of compound 5b (380 mg, 1.48 mmol) in MeCN (5 mL) wasadded(S)-4-methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide(532 mg, 0.74 mmol). The reaction mixture was stirred at 45° C.overnight. The solvent was removed under reduced pressure. The residuewas purified by flash column chromatography on silica gel(DCM/MeOH=10:1) to afford desired compound (6), which was thentransformed into the corresponding mesylate by treatment with ionexchange resin (280 mg, 39% yield); ¹H NMR (CDCl₃, 400 MHz): δ 10.15 (s,1H), 9.53 (br s, 1H), 8.03 (d, J=2.0 Hz, 1H), 7.85 (m, 1H), 7.68 (br s,1H), 7.37 (d, J=8.0 Hz, 1H), 7.26-7.13 (m, 10H), 6.84 (br s, 1H), 6.52(br s, 1H), 5.20 (m, 2H), 4.97 (m, 1H), 4.50-3.96 (m, 7H), 3.46-3.28 (m,2H), 3.16 (m, 1H), 3.06-2.92 (m, 3H), 2.85-2.61 (m, 7H), 2.44 (s, 3H),2.14 (m, 2H), 1.69-1.17 (m, 11H), 0.89-0.83 (m, 12H). Compound 5a couldalso be used for this reaction.

Example 19 was prepared from compound 7 and PEG_(5K)NHC(O)CH₂ONH₂(Creative PEGWorks, Chapel Hill, N.C., US) following general pegylationprocedure A.

Example 20:4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(3-((PEG_(5K)-imino)methyl)-4-(propionyloxy)benzyl)morpholin-4-iummethanesulfonate (7)

4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(3-formyl-4-(propionyloxy)benzyl)morpholin-4-iummethanesulfonate (6)

To a solution of compound 4 (400 mg, 1.476 mmol) in MeCN (5 mL) wasadded(S)-4-methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide(318.8 mg, 0.442 mmol). The reaction mixture was stirred at 45° C.overnight. An excess of solvent was concentrated and the residue wascrystallized repeatedly from MeCN/Et₂O (1/5, v/v) to afford the desiredcompound 5, which was transformed into the corresponding mesylate (200mg, 15% yield) by treatment with ion exchange resin; ¹H NMR (300 MHz,CDCl₃): δ 10.18 (s, 1H), 7.68 (br, s, 1H), 8.03 (s, 1H), 7.88 (m, 1H),7.72 (br, 1H), 7.38 (m, 1H), 7.30˜7.15 (m, 10H), 6.74 (m, 1H), 6.37 (br,1H), 5.2˜55.01 (m, 3H), 4.50˜3.90 (m, 12H), 3.4˜73.12 (m, 3H), 2.97 (m,2H), 2.97˜2.71 (m, 7H), 2.15 (m, 2H), 2.71˜1.10 (m, 9H), 0.87 (m, 12H).

Example 20 was prepared by methods analogous to those described inExample 16, wherein the intermediates were made in similar fashion(using ethanoyl chloride to generate the correlary to intermediate 1shown in eg 16), and compound 6 and PEG_(5K)ONH₃ ⁺.MsO⁻ followinggeneral pegylation procedure B.

Example 21:4-(3-Acetoxy-4-((PEG_(2K)-imino)methyl)benzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholin-4-iummethanesulfonate (8)

4-(4-Acetoxy-3-formylbenzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholin-4-iummethanesulfonate (7)

To a solution of compound 5b (380 mg, 1.48 mmol) in MeCN (5 mL) wasadded(S)-4-methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide(532 mg, 0.74 mmol). The reaction mixture was stirred at 45° C.overnight. The solvent was removed under reduced pressure. The residuewas purified by flash column chromatography on silica gel(DCM/MeOH=10:1) to afford desired compound (0, which was thentransformed into the corresponding mesylate by treatment with ionexchange resin (280 mg, 39% yield); ¹H NMR (CDCl₃, 400 MHz): δ 10.15 (s,1H), 9.53 (br s, 1H), 8.03 (d, J=2.0 Hz, 1H), 7.85 (m, 1H), 7.68 (br s,1H), 7.37 (d, J=8.0 Hz, 1H), 7.26-7.13 (m, 10H), 6.84 (br s, 1H), 6.52(br s, 1H), 5.20 (m, 2H), 4.97 (m, 1H), 4.50-3.96 (m, 7H), 3.46-3.28 (m,2H), 3.16 (m, 1H), 3.06-2.92 (m, 3H), 2.85-2.61 (m, 7H), 2.44 (s, 3H),2.14 (m, 2H), 1.69-1.17 (m, 11H), 0.89-0.83 (m, 12H).

Example 21 was prepared from compound 7 and PEG_(2K)ONH₃ ⁺.MsO⁻following general pegylation procedure A.

Example 22:4-(4-Acetoxy-3-(1-(PEG_(2K)-imino)ethyl)benzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholin-4-iummethanesulfonate (5)

2-Acetyl-4-methylphenyl acetate (1)

To a solution of 1-(2-hydroxy-5-methylphenyl)ethanone (1.5 g, 0.01 mol)in DCM (15 mL) were added TEA (1.5 g, 0.015 mol) and acetyl chloride(0.94 g, 0.012 mol) at 0° C. The reaction mixture was stirred at RTovernight. This mixture was quenched with water (20 mL). The DCM phasewas collected, washed with brine (20 mL), dried over anhydrous MgSO₄,and concentrated. The residue was purified by flash columnchromatography on silica gel (Petroleum Ether/EtOAc=3:1) to affordcompound 1 (0.9 g, 47% yield); ¹H NMR (300 MHz, CDCl₃): δ 7.64 (m, 1H),7.37 (m, 1H), 7.02 (d, J=8.1 Hz, 1H), 2.57 (s, 3H), 2.42 (s, 3H), 2.37(s, 3H).

2-Acetyl-4-(bromomethyl)phenyl acetate (2)

To a solution of compound 1 (0.5 g, 2.6 mmol) in CCl₄ (20 mL) were addedNBS (573 mg, 3.25 mmol) and AIBN (42.6 mg, 0.26 mmol). The reactionmixture was heated under reflux overnight. The mixture was cooled to RTand filtered. The filtrate was concentrated and the residue was purifiedby flash column chromatography on silica gel (PetroleumEther/EtOAc=10:1) to afford compound 2 (160 mg, 23% yield); ¹H NMR (300MHz, DMSO-d6): δ 8.02 (m, 1H), 7.73 (d, J=8.1 Hz, 1H), 7.25 (d, J=8.4Hz, 1H), 4.81 (s, 2H), 2.53 (s, 3H), 2.32 (s, 3H).

4-(4-Acetoxy-3-acetylbenzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholin-4-iummethanesulfonate (4)

To a solution of compound 2 (1.03 g, 3.7 mmol) in MeCN (10 mL) was addedcompound(S)-4-methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide(884.7 mg, 1.23 mmol). The reaction mixture was heated at 45° C.overnight. An excess of solvent was concentrated and the residue waspurified by flash column chromatography on silica gel (EtOAc/MeOH=100:3)to afford the desired compound 3, which was transformed into thecorresponding mesylate (260 mg, 21% yield) by treatment with ionexchange resin; ¹H NMR (300 MHz, CDCl₃): δ 9.62 (m, 1H), 8.06 (m, 1H),7.88˜7.71 (m, 2H), 7.33˜7.11 (m, 11H), 6.95 (m, 1H), 6.66 (m, 1H),5.33˜4.91 (m, 2H), 4.55˜3.90 (m, 11H), 3.5˜82.91 (m, 4H), 2.85 (s, 3H),2.74 (m, 2H), 2.61 (s, 3H), 2.40 (s, 3H), 2.31˜1.94 (m, 7H), 1.72˜1.18(m, 8H), 0.88 (m, 12H).

Example 22 was prepared from compound 4 and PEG_(2K)ONH₃ ⁺.MsO⁻following general pegylation procedure B.

Example 23:4-(3-Acetoxy-4-((PEG_(3K)-imino)methyl)benzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholin-4-iummethanesulfonate (8)

4-(4-Acetoxy-3-formylbenzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholin-4-iummethanesulfonate (7)

To a solution of compound 5b (380 mg, 1.48 mmol) in MeCN (5 mL) wasadded(S)-4-methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide(532 mg, 0.74 mmol). The reaction mixture was stirred at 45° C.overnight. The solvent was removed under reduced pressure. The residuewas purified by flash column chromatography on silica gel(DCM/MeOH=10:1) to afford desired compound (0, which was thentransformed into the corresponding mesylate by treatment with ionexchange resin (280 mg, 39% yield); ¹H NMR (CDCl₃, 400 MHz): δ 10.15 (s,1H), 9.53 (br s, 1H), 8.03 (d, J=2.0 Hz, 1H), 7.85 (m, 1H), 7.68 (br s,1H), 7.37 (d, J=8.0 Hz, 1H), 7.26-7.13 (m, 10H), 6.84 (br s, 1H), 6.52(br s, 1H), 5.20 (m, 2H), 4.97 (m, 1H), 4.50-3.96 (m, 7H), 3.46-3.28 (m,2H), 3.16 (m, 1H), 3.06-2.92 (m, 3H), 2.85-2.61 (m, 7H), 2.44 (s, 3H),2.14 (m, 2H), 1.69-1.17 (m, 11H), 0.89-0.83 (m, 12H).

Example 23 was prepared by methods analogous to those described inExample 16, wherein the intermediates were made in similar fashion(using acetyl chloride to generate the corollary intermediate 1 shown ineg 16), and compound 7 and PEG_(3K)ONH₃ ⁺.MsO⁻ following generalpegylation procedure A.

Example 24:4-(4-Acetoxy-3-(1-(PEG_(3K)-imino)ethyl)benzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholin-4-iummethanesulfonate (5)

4-(4-Acetoxy-3-acetylbenzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholin-4-iummethanesulfonate (4)

To a solution of compound 2 (1.03 g, 3.7 mmol) in MeCN (10 mL) was addedcompound(S)-4-methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide(884.7 mg, 1.23 mmol). The reaction mixture was heated at 45° C.overnight. An excess of solvent was concentrated and the residue waspurified by flash column chromatography on silica gel (EtOAc/MeOH=100:3)to afford the desired compound 3, which was transformed into thecorresponding mesylate (260 mg, 21% yield) by treatment with ionexchange resin; ¹H NMR (300 MHz, CDCl₃): δ 9.62 (m, 1H), 8.06 (m, 1H),7.88˜7.71 (m, 2H), 7.33˜7.11 (m, 11H), 6.95 (m, 1H), 6.66 (m, 1H),5.33˜4.91 (m, 2H), 4.55˜3.90 (m, 11H), 3.58˜2.91 (m, 4H), 2.85 (s, 3H),2.74 (m, 2H), 2.61 (s, 3H), 2.40 (s, 3H), 2.31˜1.94 (m, 7H), 1.72˜1.18(m, 8H), 0.88 (m, 12H).

Example 24 was prepared by methods analogous to those described inExample 22, wherein the intermediates were made in similar fashion andcompound 4 and PEG_(3K)ONH₃ ⁺.MsO⁻ following general pegylationprocedure B.

Example 25.4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(3-(2-(PEG_(20K)-4-Arm-imino)ethoxy)-4-(pivaloyloxy)benzyl)morpholin-4-iummethanesulfonate

Example 25 was prepared by methods analogous to those described inExample 17, wherein the intermediates were made in similar fashion,while using PEG_(20K)ONH₃ ⁺.MsO⁻ and following general pegylationprocedure B.

Example 26:4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(4-(isobutyryloxy)-3-((PEG_(5K)-imino)methyl)benzyl)morpholin-4-iummethanesulfonate (6)

4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(3-formyl-4-(isobutyryloxy)benzyl)morpholin-4-iummethanesulfonate (5)

To a solution of compound 3 (550 mg, 1.657 mmol) in MeCN (8 mL) wasadded(S)-4-methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide(393 mg, 0.547 mmol). The reaction mixture was stirred at 40° C.overnight. An excess of solvent was concentrated and the residue wascrystallized repeatedly from (EtOAc/Et₂O=1:5) to afford the desiredcompound 4, which was transformed into the corresponding mesylate 5 (115mg, 7.5% yield) by treatment with ion exchange resin; ¹H NMR (400 MHz,CDCl₃): δ 10.18 (s, 1H), 9.68 (m, 1H), 8.04 (m, 1H), 7.89 (m, 1H), 7.81(s, 1H), 7.35 (m, 1H), 7.30 (m, 1H), 7.11-7.29 (m, 9H), 6.79 (s, 1H),6.44 (m, 1H), 5.18 (m, 2H), 4.99 (m, 1H), 4.41 (m, 3H), 4.20 (m, 3H),3.99 (m, 3H), 3.40 (m, 1H), 3.30 (m, 1H), 3.20 (m, 1H), 2.95 (m, 2H),2.92 (m, 1H), 2.79 (m, 3H), 2.75 (m, 2H), 2.21 (m, 1H), 2.09 (m, 1H),1.83 (m, 4H), 1.62 (m, 2H), 1.49 (m, 4H), 1.38 (m, 6H), 1.24 (m, 2H),0.88 (m, 12H).

Example 26 was prepared by methods analogous to those described inExample 16, wherein the intermediates were made in similar fashion(using isopropanoyl chloride to generate the corollary intermediate 1shown in eg 16), and compound 5 and PEG_(5K)ONH₃ ⁺.MsO⁻ followinggeneral pegylation procedure B.

Example 27:4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(3-((PEG_(5K)-imino)methyl)-4-(pivaloyloxy)benzyl)morpholin-4-iummethanesulfonate (6)

4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(3-formyl-4-(pivaloyloxy)benzyl)morpholin-4-iummethanesulfonate (5)

To a solution of compound 3 (500 mg, 1.44 mmol) in MeCN (8 mL) was added(S)-4-methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide(360 mg, 0.5 mmol). The reaction mixture was stirred at 40° C.overnight. An excess of solvent was concentrated and the residue wascrystallized repeatedly from (EtOAc/Et₂O=1:5) to afford the desiredcompound 4, which was transformed into the corresponding mesylate 5 (130mg, 12% yield) by treatment with ion exchange resin; ¹H NMR (400 MHz,CDCl₃): δ 10.17 (s, 1H), 9.74 (m, 1H), 8.01 (m, 1H), 7.90 (m, 1H), 7.74(m, 1H), 7.36-7.12 (m, 11H), 6.78 (m, 1H), 6.41 (m, 1H), 5.22 (m, 1H),5.14 (m, 2H), 4.58-4.35 (m, 3H), 4.28-4.10 (m, 3H), 4.08-3.83 (m, 3H),3.38 (m, 1H), 3.29 (m, 1H), 3.17 (m, 1H), 2.97 (m, 2H), 2.83 (s, 3H),2.76 (m, 2H), 2.30-2.20 (m, 2H), 1.70-1.58 (m, 2H), 1.47 (m, 6H), 1.42(s, 10H), 1.30-1.16 (m, 3H), 0.90-0.84 (m, 12H).

Example 27 was prepared by methods analogous to those described inExample 16, wherein the intermediates were made in similar fashion(using t-butanoyl chloride to generate the corollary intermediate 1shown in eg 16), and compound 5 and PEG_(5K)ONH₃ ⁺.MsO⁻ followinggeneral pegylation procedure B.

Example 28:4-(4-Acetoxy-3-(1-(PEG_(5K)-imino)ethyl)-5-methylbenzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholin-4-iummethanesulfonate (7)

2-Hydroxy-5-(hydroxymethyl)-3-methylbenzaldehyde (1)

To a mixture of 2-hydroxy-3-methylbenzaldehyde (5.01 g, 36.84 mmol) andformaldehyde (37%, 7.01 g, 86.45 mmol) was added concentrated HCl (30mL) at RT. The reaction mixture was heated at 80° C. for 1 h. Water (90mL) was added and the resulting mixture was extracted with EtOAc (100mL×3). The combined organic phases were concentrated and the residue wastreated with water (150 mL, 40° C.). The solid was filtered off and thefiltrate was extracted with EtOAc (100 mL×3). The combined organicphases were dried over anhydrous MgSO₄ and concentrated to affordcompound 1 (2.60 g, 43% yield); ¹H NMR (400 MHz, CDCl₃): δ 11.26 (s,1H), 9.88 (s, 1H), 7.41 (s, 2H), 4.66 (s, 2H), 2.28 (s, 3H).

5-(((tert-Butyldimethylsilyl)oxy)methyl)-2-hydroxy-3-methylbenzaldehyde(2)

To a solution of compound 1 (2.60 g, 15.66 mmol) in DCM (50 mL) wasadded imidazole (2.13 g, 31.33 mmol) at 0° C. A solution of TBSCl (3.54g, 23.50 mmol) in DCM (5 mL) was added and the reaction mixture wasstirred at room temperature for 1 h. The mixture was concentrated andthe residue was purified by flash column chromatography on silica gel(Hexane/EtOAc=200:1) to give compound 2 (3.90 g, 88.9% yield); ¹H NMR(400 MHz, CDCl₃): δ 10.85 (br, s, 1H), 9.96 (s, 1H), 7.44 (d, J=1.6 Hz,1H), 7.33 (d, J=1.2 Hz, 1H), 4.56 (s, 2H), 2.12 (s, 3H), 0.82 (s, 9H),0.00 (s, 6H).

4-(((tert-Butyldimethylsilyl)oxy)methyl)-2-formyl-6-methylphenyl acetate(3)

To a solution of compound 2 (1.70 g, 6.08 mmol) in DCM (50 mL) wereadded TEA (1.23 g, 12.14 mmol) and acetyl chloride (715 mg, 9.11 mmol)at 0° C. The reaction mixture was stirred at RT for 20 min. The mixturewas diluted with DCM (50 mL) and then poured into water (100 mL). Thetwo phases were separated and the organic phase was washed with brine(100 mL), dried over anhydrous MgSO₄ and concentrated to afford crudecompound 3 (1.94 g, quantitative), which was used in the next stepwithout further purification; ¹H NMR J400 MHz, CDCl₃): δ 9.90 (s, 1H),7.52 (d, J=1.6 Hz, 1H), 7.35 (d, J=1.2 Hz, 1H), 4.63 (s, 2H), 2.30 (s,3H), 2.11 (s, 3H), 0.83 (s, 9H), 0.00 (s, 6H).

2-Formyl-4-(iodomethyl)-6-methylphenyl acetate (4)

To a solution of NaI (4.66 g, 31.06 mmol) in MeCN (50 mL) were addedcompound 3 (2.01 g, 6.21 mmol) and SiCl₄ (1.06 g, 6.21 mmol) at 0° C.The reaction mixture was stirred for 15 min at RT. The mixture wasconcentrated and the residue was treated with DCM (100 mL). Theresulting mixture was filtered and the filtrate was washed withsaturated Na₂S₂O₃ (50 mL×2), dried over anhydrous MgSO₄ andconcentrated. The residue was purified by flash column chromatography onsilica gel (Hexane/EtOAc=15:1) to give compound 4 (803 mg, 41% yield);¹H NMR (400 MHz, CDCl₃): δ 10.00 (s, 1H), 7.70 (d, J=2.4 Hz, 1H), 7.52(d, J=2.0 Hz, 1H), 4.44 (s, 2H), 2.42 (s, 3H), 2.22 (s, 3H).

4-(4-Acetoxy-3-acetyl-5-methylbenzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholin-4-iummethanesulfonate (6)

To a solution of compound 4 (803 mg, 1.57 mmol) in MeCN (5 mL) was added(S)-4-methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide(340 mg, 0.47 mmol). The reaction mixture was stirred at 40° C.overnight. An excess of solvent was concentrated and the residue wasrecrystallized three times from (EtOAc/Et₂O=1:5) to afford the desirediodide salt 5, which was transformed into the corresponding mesylate 6(202 mg, 47% yield) by treatment with ion exchange resin; ¹H NMR (400MHz, CDCl₃): δ 10.05 (s, 1H), 9.55 (m, 1H), 7.86 (m, 1H), 7.74 (m, 2H),7.13-7.29 (m, 10H), 6.85 (m, 1H), 6.51 (m, 1H), 5.23 (m, 1H), 5.05 (m,2H), 4.45 (m, 5H), 4.22 (m, 5H), 4.00 (m, 4H), 3.30-3.52 (m, 2H), 3.17(m, 1H), 2.98 (m, 2H), 2.83 (s, 4H), 2.76 (m, 2H), 2.46 (s, 3H), 2.29(s, 3H), 2.08-2.25 (m, 2H), 1.48-1.69 (m, 4H), 1.45 (m, 2H), 1.38 (m,2H), 1.25 (m, 2H), 0.88 (m, 12H).

Example 28 was prepared from compound 6 and PEG_(5K)ONH₃ ⁺.MsO⁻following general pegylation procedure B.

Example 29:4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(3-((PEG_(20K)-4-Arm-imino)methyl)-4-(pivaloyloxy)benzyl)morpholin-4-iummethanesulfonate

4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(3-formyl-4-(pivaloyloxy)benzyl)morpholin-4-iummethanesulfonate (5)

To a solution of compound 3 (500 mg, 1.44 mmol) in MeCN (8 mL) was added(S)-4-methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide(360 mg, 0.5 mmol). The reaction mixture was stirred at 40° C.overnight. An excess of solvent was concentrated and the residue wascrystallized repeatedly from (EtOAc/Et₂O=1:5) to afford the desiredcompound 4, which was transformed into the corresponding mesylate 5 (130mg, 12% yield) by treatment with ion exchange resin; ¹H NMR (400 MHz,CDCl₃): δ 10.17 (s, 1H), 9.74 (m, 1H), 8.01 (m, 1H), 7.90 (m, 1H), 7.74(m, 1H), 7.36-7.12 (m, 11H), 6.78 (m, 1H), 6.41 (m, 1H), 5.22 (m, 1H),5.14 (m, 2H), 4.58-4.35 (m, 3H), 4.28-4.10 (m, 3H), 4.08-3.83 (m, 3H),3.38 (m, 1H), 3.29 (m, 1H), 3.17 (m, 1H), 2.97 (m, 2H), 2.83 (s, 3H),2.76 (m, 2H), 2.30-2.20 (m, 2H), 1.70-1.58 (m, 2H), 1.47 (m, 6H), 1.42(s, 10H), 1.30-1.16 (m, 3H), 0.90-0.84 (m, 12H).

Example 29 was prepared by methods analogous to those described inExample 18, wherein the intermediates were made in similar fashion(using t-butanoyl chloride to generate the corollary intermediate 1shown in eg 18), and compound 5 and PEG_(20K)-(ONH₃ ⁺.MsO⁻)₄ followinggeneral pegylation procedure B.

Example 30:4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(3-(PEG_(20K)-4-Arm-imino)methyl)-5-methyl-4-(pivaloyloxy)benzyl)morpholin-4-iummethanesulfonate (6)

4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(3-formyl-5-methyl-4-(pivaloyloxy)benzyl)morpholin-4-iummethanesulfonate (5)

To a solution of compound 3 (900 mg, 2.60 mmol) in MeCN (8 mL) was added(S)-4-methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide(624 mg, 0.87 mmol). The reaction mixture was stirred at 40° C.overnight. An excess of solvent was concentrated and the residue wascrystallized repeatedly from (EtOAc/Et₂O=1:5) to afford the desiredcompound 4, which was transformed into the corresponding mesylate 5 (222mg, 9.0% yield) by treatment with ion exchange resin; ¹H NMR (400 MHz,CDCl₃): δ 10.05 (s, 1H), 9.66 (m, 1H), 7.82 (m, 3H), 7.12-7.30 (m, 10H),6.84 (m, 1H), 6.47 (m, 1H), 5.19 (m, 1H), 5.01 (m, 2H), 4.47 (m, 5H),4.20 (m, 4H), 3.98 (m, 4H), 3.40 (m, 1H), 3.28 (m, 1H), 3.17 (m, 1H),2.98 (m, 2H), 2.83 (m, 4H), 2.73 (m, 2H), 2.25 (m, 3H), 2.10 (m, 2H),1.61 (m, 2H), 1.47 (m, 12H), 1.26 (m, 2H), 0.87 (m, 12H).

Example 30 was prepared by methods analogous to those described inExample 28, wherein the intermediates were made in similar fashion(using t-butanoyl chloride to generate the corollary intermediate 1shown in eg 28), and compound 5 and PEG_(20K)(ONH₃ ⁺.MsO⁻)₄ followinggeneral pegylation procedure B.

Example 31:4-(4-Acetoxy-3-((4-(PEG-imino)methyl)benzyl)oxy)benzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholin-4-iummethanesulfonate (8)

1-(Chloromethyl)-4-(dimethoxymethyl)benzene (1)

To a solution of (4-(dimethoxymethyl)phenyl)methanol (200 mg, 1.1 mmol)in DCM (10 ml) were added TEA (365.6 mg, 3.62 mmol) and MsCl (207.6 mg,1.813 mmol) at 0° C. The reaction mixture was stirred at RT for 2 h, andthen poured into saturated NaHCO₃ (10 mL). The two phases wereseparated, and the organic layer was dried over anhydrous Na₂SO₄ andconcentrated to afford compound 1 (200 mg, 91%), which was used in thenext step without further purification.

4-(4-(Dimethoxymethyl)benzyloxy)-3-hydroxybenzaldehyde (2)

To a solution of compound 1 (200 mg, 0.998 mmol) in DMSO (5 mL) wasadded NaH (37.4 mg, 1.1 mmol) at room temperature. After 30 min ofreaction, a solution of 3,4-dihydroxybenzaldehyde (137.7 mg, 0.998 mmol)in DMSO (5 mL) was added and the reaction mixture was stirred at RTovernight. The mixture was poured into saturated NaHCO₃ (10 mL) and theresulting mixture was extracted with DCM (10 mL×2). The combined organiclayers were dried over anhydrous Na₂SO₄ and concentrated to affordcompound 2 (300 mg, crude), which was used in the next step withoutfurther purification.

2-(4-(Dimethoxymethyl)benzyloxy)-4-formylphenyl acetate (3)

To a solution of compound 2 (300 mg, 1 mmol) in DCM (10 mL) were addedTEA (202 mg, 2 mmol) and AcCl (102 mg, 1.3 mmol) at 0° C. The reactionmixture was stirred at r.t. for 2 h, and then poured into saturatedNaHCO₃ (10 mL). The two phases were separated, and the organic layer wasdried over anhydrous Na₂SO₄ and concentrated to afford compound 3 (200mg, crude), which was used in the next step without furtherpurification.

2-(4-(Dimethoxymethyl)benzyloxy)-4-(hydroxymethyl)phenyl acetate (4)

To a solution of compound 3 (200 mg, 0.58 mmol) in DCM/MeOH (10 mL/1mL), was added NaBH₄ (19.8 mg, 0.58 mmol). The reaction mixture wasstirred at 0° C. for 1 hour and then quenched with acetone (5 mL). Themixture was poured into saturated aq. NaHCO₃ (10 mL) and extracted withDCM (10 mL×2). The combined organic layers were dried over anhydrousNa₂SO₄ and concentrated to afford compound 4 (300 mg, crude), which wasused in next step without further purification.

4-(Bromomethyl)-2-(4-formylbenzyloxy)phenyl acetate (5)

To a solution of compound 4 (1.8 g, 5.2 mmol) in DCM (50 mL) was addedPBr₃ (1.41 g, 5.2 mmol). The reaction mixture was stirred at RT for 5hours and then quenched with saturated aq. NaHCO₃ (60 mL). The twolayers were separated and the aqueous phase was extracted with DCM (50mL×2). The combined organic layers were dried over anhydrous Na₂SO₄ andconcentrated. The residue was purified by flash column chromatography onsilica gel (Petroleum Ether/EtOAc=4:1) to afford compound 5 (327 mg, 15%yield); ¹H NMR (400 MHz, DMSO): δ10.01 (s, 1H), 7.96˜7.94 (m, 2H),7.6˜27.60 (m, 2H), 7.31 (m, 1H), 7.13˜7.07 (m, 2H), 5.26 (s, 2H), 4.68(s, 2H), 2.27 (s, 3H).

4-(4-Acetoxy-3((4-formylbenzyl)oxy)benzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholin-4-iummethanesulfonate (7)

To a solution of compound 5 (320 mg, 0.884 mmol) in MeCN (6 mL) wasadded(S)-4-methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide(254 mg, 0.354 mmol). The reaction mixture was stirred at 45° C. for 48hours. An excessive solvent was evaporated and the residue wasrepeatedly crystallized from MeCN/Et₂O (1/5, v/v) to afford the desiredproduct 6, which was then transformed into the corresponding mesylatecompound 7 (180 mg, 47% yield) by treatment with ion exchange resin.

A solution of compound 7 (500 mg, 0.46 mmol), 2-amino-5-methoxybenzoicacid (25.5 mg, 0.14 mmol) and PEG-O—NH₂ (mesylate salt, 2.12 g, 0.41mmol) in DCM was stirred at r.t. for 2 h. The reaction mixture was thenconcentrated and the residue was dissolved in/PrOH at 40° C. Thesolution was cooled to RT and Et₂O was added to induce crystallization.The mixture was kept in ice bath for 10 min and then filtered. Thefiltration cake was crystallized from ^(i)PrOH/Et₂O (5/2) to afford 8(2.10 g, 82% yield).

Example 32:4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(4-(isobutyryloxy)-3-((4-(PEG_(5K)-imino)methyl)benzyl)oxy)benzyl)morpholin-4-iummethanesulfonate (8)

4-((4S,7S,10S,13S)-10-Benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(3-(4-formylbenzyloxy)-4-(isobutyryloxy)benzyl)morpholin-4-iummethanesulfonate (7)

To a solution of compound 5 (310.4 mg, 0.80 mmol) in MeCN (2 mL) wasadded(S)-4-methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide(286 mg, 0.30 mmol). The reaction mixture was stirred at 45° C. for 48hours. An excessive solvent was evaporated and the residue wasrepeatedly crystallized from MeCN/Et₂O (1/5, v/v) to afford the desiredproduct 6, which was then transformed into the corresponding mesylatecompound 7 (280 mg, 83% yield) by treatment with ion exchange resin.

A solution of compound 6 (280 mg, 0.25 mmol), 2-amino-5-methoxybenzoicacid (14.0 mg, 0.026 mmol) and PEG-O—NH₂ (mesylate salt, 1.16 g, 0.227mmol) in DCM (3 mL) was stirred at r.t. for 2 h. The reaction mixturewas then concentrated and the residue was dissolved in i-PrOH at 40° C.The solution was cooled to room temperature and Et₂O was added to inducecrystallization. The mixture was kept in ice bath for 10 min and formedsolid was collected by filtration. Crystallization from i-PrOH/Et₂O(5:2) was repeated twice until all 7 was removed to afford 8 (1.0 g, 72%yield).

Example 33:4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(3-((4-(PEG_(5K)-imino)methyl)benzyl)oxy)-4-(pivaloyloxy)benzyl)morpholin-4-iummethanesulfonate (8)

4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)-4-(3-(4-formylbenzyloxy)-4-(pivaloyloxy)benzyl)morpholin-4-iummethanesulfonate (7)

To a solution of compound 5 (t-butyl ester analog of compound 5 inexample 31, 230 mg, 0.51 mmol) in MeCN (3 mL) was added(S)-4-methyl-N—((S)-1-(((S)-4-methyl-1-((R)-2-methyloxiran-2-yl)-1-oxopentan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)-4-phenylbutanamido)pentanamide(184 mg, 0.25 mmol). The reaction mixture was stirred at 45° C. for 48hours. An excessive solvent was evaporated and the residue wasrepeatedly crystallized from MeCN/Et₂O (1/5, v/v) to afford the desiredproduct 6, which was then transformed into the corresponding mesylatesalt compound 7 (170 mg, 71% yield) by treatment with ion exchangeresin; ¹H NMR (400 MHz, CDCl₃): δ 9.99 (s, 1H), 9.59 (m, 1H), 7.87˜7.85(m, 2H), 7.57˜7.55 (m, 2H), 7.32 (m, 1H), 7.26˜7.13 (m, 11H), 7.03 (m,1H), 6.89 (m, 1H), 6.44 (m, 1H), 5.18 (m, 2H), 5.05˜5.03 (m, 1H),4.92˜4.88 (m, 2H), 4.46˜4.42 (m, 4H), 4.18˜4.03 (m, 4H), 3.94˜3.90 (m,3H), 3.40˜3.32 (m, 1H), 3.27˜3.20 (m, 1H), 3.13 (m, 1H), 3.00˜2.98 (m,2H), 3.82˜2.80 (m, 3H), 2.79 (m, 1H), 2.74˜2.72 (m, 2H), 2.19 (m, 1H),2.10˜1.97 (m, 4H), 1.64˜1.55 (m, 2H), 1.48˜1.44 (m, 5H), 1.29 (m, 6H),0.88˜0.81 (m, 12H).

A solution of compound 7 (170 mg, 0.15 mmol), 2-amino-5-methoxybenzoicacid (28.2 mg, 0.016 mmol) and PEG-O—NH2 (mesylate salt, 614 mg, 0.12mmol) in DCM was stirred at RT for 30 min. The reaction mixture was thenconcentrated and the residue was dissolved in ^(i)PrOH at 40° C. Thesolution was cooled to RT and Et₂O was added to induce crystallization.The mixture was kept in ice bath for 10 min and then filtered. Thefiltration cake was crystallized from PrOH/Et₂O (5/2) to afford 8 (580mg, 77%).

Representative carfilzomib prodrug examples 31-33 of the inventionprovide oxime linked conjugates with potentially enhanced chemicalstability. In these examples the oxime linkage is spaced with anelectron donating benzyloxy group to increase the overall stability ofthe PEG construct.

Example 34:4-(4-Acetoxy-3-((1-PEG_(3K)-1H-1,2,3-triazol-4-yl)methoxy)benzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholin-4-iumchloride

Example 34 was prepared using a method analogous to that taught inExamples 5-11 and Method A, but using the chloride salt intermediatehaving a chloride anion as the counter ion.

Example 35:4-(4-Acetoxy-3-((1-PEG_(3K)-1H-1,2,3-triazol-4-yl)methoxy)benzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholin-4-iummesylate

Example 35 was prepared using a method analogous to that taught inExamples 5-11 and Method A using a PEG_(3K)N₃. ¹H NMR (DMSO-d6, 400MHz): δ 9.19 (M, 1H), 8.24 (m, 2H), 8.12 (m, 1H), 7.90 (m, 1H), 7.62 (m,1H), 7.22 (m, 13H), 7.0 (m, 1H), 5.26 (m, 2H), 4.88 (m, 2H), 4.53 (m,3H), 4.37 (br s, 4H), 4.05 (m, 5H), 3.81 (m, 2H), 3.68 (m, 4H), 3.52 (brs, 339H), 3.30 (m, 4H), 3.24 (s, 4H), 2.94 (m, 2H), 2.75 (m, 1H), 2.63(m, 2H), 2.24 (s, 3H), 1.87 (m, 2H), 1.59 (m, 2H), 1.40 (m, 7H), 0.84(m, 12H)

Example 36:4-(4-Acetoxy-3-((1-PEG_(2K)-1H-1,2,3-triazol-4-yl)methoxy)benzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholin-4-iummesylate

Example 36 was prepared using a method analogous to that taught inExamples 5-11 and Method A using a PEG_(2K)N₃. ¹H NMR (DMSO-d6, 400MHz): δ 9.44 (M, 1H), 8.26 (m, 2H), 8.16 (m, 1H), 8.00 (m, 1H), 7.62 (m,1H), 7.22 (m, 13H), 5.26 (m, 2H), 5.00 (m, 2H), 4.54 (m, 3H), 4.37 (m,5H), 4.09 (m, 4H), 3.81 (m, 2H), 3.68 (m, 2H), 3.50 (br s, 218H), 3.32(m, 2H), 3.27 (s, 1H), 3.26 (s, 4H), 2.94 (m, 2H), 2.76 (m, 1H), 2.61(m, 2H), 2.24 (s, 3H), 1.90 (m, 2H), 1.62 (m, 2H), 1.40 (m, 7H), 0.82(m, 12H)

Example 37:4-(4-acetoxy-3-((1-(5-((2-PEG_(3K)-ethyl)amino)-5-oxopentyl)-1H-1,2,3-triazol-4-yl)methoxy)benzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholin-4-iummesylate

Example 37 was prepared using a method analogous to that taught inExamples 5-11 and Method A using a PEG_(3K) with a linker derived from5-azidopentanoic acid. ¹H NMR (DMSO-d6, 400 MHz): δ 9.26 (M, 1H), 8.25(m, 2H), 8.17 (m, 1H), 7.98 (d, 1H), 7.87 (m, 1H), 7.62 (m, 1H), 7.22(m, 11H), 7.02 (m, 1H), 4.99 (m, 2H), 4.56 (m, 1H), 4.37 (br s, 6H),4.11 (m, 3H), 3.71 (m, 3H), 3.52 (br s, 304H), 3.25 (br s, 7H), 2.75 (m,1H), 2.61 (m, 2H), 2.24 (s, 3H), 2.11 (m, 1H), 1.87 (m, 2H), 1.40 (m,5H), 0.82 (m, 12H)

Example 38:4-(4-acetoxy-3-((1-(5-((2-PEG_(2K)-ethyl)amino)-5-oxopentyl)-1H-1,2,3-triazol-4-yl)methoxy)benzyl)-4-((4S,7S,10S,13S)-10-benzyl-7-isobutyl-15-methyl-13-((R)-2-methyloxirane-2-carbonyl)-2,5,8,11-tetraoxo-4-phenethyl-3,6,9,12-tetraazahexadecyl)morpholin-4-iummesylate

Example 38 was prepared using a method analogous to that taught inExamples 5-11 and Method A using a PEG_(2K) with a linker derived from5-azidopentanoic acid. ¹H NMR (DMSO-d6, 400 MHz): δ 9.56 (M, 1H), 8.29(m, 2H), 8.18 (m, 1H), 8.04 (m, 1H), 7.92 (m, 1H), 7.62 (m, 1H), 7.22(m, 11H), 7.0 (m, 1H), 5.38 (m, 2H), 4.99 (m, 2H), 4.56 (m, 1H), 4.37(br s, 7H), 4.19 (m, 3H), 4.02 (m, 3H), 3.52 (br s, 179H), 3.25 (br s,5H), 2.94 (m, 2H), 2.80 (m, 1H), 2.63 (m, 2H), 2.24 (s, 3H), 2.10 (m,2H), 1.87 (m, 2H), 1.40 (m, 9H), 0.82 (m, 12H)

The Exemplary compounds of the present invention may be shown to beeffective in treating various cancers, including without limitation,multiple myeloma, by virtue of possessing pharmacokinetic andpharmacodynamic profiles suitable and sufficient to provide such cancertreatment. The following exemplary descriptions and accompanying figuresshow some of these pharmacokinetic and phamacodynamic profiles of selectrepresentative compounds of the invention.

Example 39: PEG-Carfilzomib Compound Conversion in Human Plasma HumanPlasma Conversion Protocol:

A 1 millimolar (mM) stock of the desired test compound was prepared inDMSO. 25 μM stock of the test compound was prepared inacetonitrile:water by diluting from 1 mM stock (i.e. 2.5 μL of 1 mMstock solution was added to 97.5 μL of acetonitrile:water (50:50)). Thefrozen human plasma (pooled from 5 males, 2KEDTA anticoagulant) wasthawed at room temperature and centrifuged at 1400×RCF 4° C., for 15minutes. Approximately 90% of the clear supernatant fraction wastransferred to a separate tube and was used for the assay. For time 0min samples, plasma was heat inactivated at 80° C. To 72 μL of heatinactivated plasma, 3 μL of 25 μM working stock was added and 50 μL ofsample was crashed with 200 μL of acetonitrile containing internalstandard. For assay, 1 μM incubation sample was prepared by spiking 20μL of 25 μM working stock to 480 μL of plasma. Samples were incubatedfor 0.5, 1, 2, 4 and 6 h at 37° C. in shaker water bath with gentleshaking. At each time point, 50 μL sample was precipitated with 200 μLof acetonitrile containing internal standard and centrifuged at4000×RCF, 4° C. for 20 minutes. 150 μL of supernatant was diluted with150 μL of water and analyzed by LC-MS/MS. An 8 point calibration curvewas generated using plasma with 5 μM highest concentration ofcarfilzomib followed by 2.5 fold dilution. The amount of carfilzomibreleased was quantified against the calibration curve and reported inμM.

FIG. 1 shows the rate of conversion of representative examples ofPEG-carfilzomib compounds to free, non-PEG conjugated, active form ofcarfilzomib. As shown, the exemplified compounds of the inventionprovide plasma concentrations of carfilzomib beginning at time 0 andgradually increasing, for most of the examples depicted, to meaningfulconcentrations for as long as 2 hours and, in some instance, for longerthan 2 hours. This figure shows that the half life of exemplarycompounds of the present invention is projected to be at least 2 hours,and potentially longer in human plasma. Thus, FIG. 1 illustrates thatPEG-carfilzomib compounds of the present invention provide a slowrelease of the active form of carfilzomib into the blood plasma, therebyallowing carfilzomib a potentially longer duration of action on cellularproteosome enzymes resulting in an expected prolonged inhibitory effecton proteosome activity.

Mean plasma concentration (μM) following intravenous administration ofPEG-carfilzomib conjugate to female Balb/c mice (n=3). Dosage indicatedis mg/kg of PEG-carfilzomib conjugate.

Example 40: PEG-Carfilzomib Mouse pK

PEG carfilzomib compounds were administered to mice (Balb/c, female, n=3per dose group) as an intravenous (i.v.) bolus at the specified dose(dose volume 5 mL/kg) in an aqueous solution containing 10% (w/v)ethanol. Blood samples were collected at the indicated time points andplasma carfilzomib concentrations were measured in duplicate byLC/MS-MS. The control comparator profile was a carfilzomib standardformulation, i.e., an aqueous solution formulation of 10% (w/v)sulfobutylether-β-cyclodextrin and 10 mmol/L sodium citrate (pH 3.5) foradministration (5 mg/kg). This standard carfilzomib represents thecurrently approved formulation for carfilzomib for treatment of multiplemyeloma. The different doses administered to the mice for examples 13,16 and 18 reflect an amount of carfilzomib present and dosed, and anamount calculated to be approximately the same as that amount ofcarfilzomib provided in the standard carfilzomib formulation.

As depicted in FIG. 2, while example 18 exhibited a profile similar tothat of the control carfilzomib, Examples 13 and 16 exhibited extensionsin their profiles. Particularly, Example 16 possessed improvedavailability of free, active carfilzomib over the same time period asthat of the control. However, Example 13 exhibited a carfilzomib releaseover a far longer period of time than the control carfilzomib, whichresulted in a significantly higher concentration of the carfilzomib theplasma over that longer period of time. The plasma concentration ofExample 13 was multiple log-fold over that of the control.

Example 41: Proteasome Inhibition of PEG-Carfilzomib Compounds Vs.Carfilzomib

PEG carfilzomib compound example 1 was administered to mice (Balb/c,female, n=3 per dose group) as an intravenous (i.v.) bolus at thespecified dose (dose volume 5 mL/kg) in an aqueous solution containing10% (w/v) ethanol. Carfilzomib standard was formulated in an aqueoussolution of 10% (w/v)sulfobutylether-β-cyclodextrin and 10 mmol/L sodiumcitrate (pH 3.5) for administration (5 mg/kg). At selected time pointsafter i.v. drug administration, tissue samples (adrenal, heart, liverand bone marrow) were collected. Whole blood was collected by cardiacpuncture into tubes containing sodium heparin.

Blood:

Approximately 0.4 mL of whole blood is collected using either EDTAmicrocentrifuged tubes. Samples are immediately placed on ice and spunat maximum speed for 2 minutes in a microcentrifuge at room temperature(RT). Cell pellets are stored on wet ice. Whole blood cell pellets areresuspended in 1 ml of phosphate buffered saline (PBS) and centrifugedat max speed at 4° C. Supernatants are removed and the pellets arewashed a second time with PBS. Samples are resuspended in 2 volumes oflysis buffer (20 mM Tris, pH 8.0, 5 mM EDTA) then frozen and stored at−80° C. until analysis.

Adrenal Glands, Heart and Liver Tissue:

Tissues (adrenal glands, heart, and liver) were collected at the graphictime points specified after dosing. Tissues were excised and placed into15 ml tubes containing PBS at 4° C. For tissues which were homogenized,samples were minced with scissors and ˜0.1-0.2 mg portions were placedinto 2 mL microcentrifuge tubes. Tissue portions were frozen and storedat −80° C.

Sample Processing:

All samples were thawed on ice. All single cell pellets in lysis buffer(whole blood) were briefly vortexed then spun at 14,000 rpm in amicrocentrifuge at 4° C. for 15 minutes. Supernatant was transferred ata ratio of 100 μL to 25 μL of 50% glycerol in a sample plate for a finalconcentration of 10% glycerol. These samples were then ready forassaying, or they may be frozen at −80° C. Approximately 2 volumes oflysis buffer and a stainless steel bead were added to the thawed tissueportions (adrenal glands, heart, and liver). Samples were homogenized at20 mHz for 60 seconds on each side then spun at 14,000 rpm in amicrocentrifuge at 4° C. for 15 minutes. Supernatant was transferred ata ratio of 100 μL to 25 μL of 50% glycerol in a sample plate for a finalconcentration of 10% glycerol. Care was taken to avoid the top lipidlayer for tissues with high fat content (i.e. adrenal). These sampleswere then ready for assaying, or they may be frozen at −80° C. Samplesfrozen at this lysate/10% glycerol stage should be thawed on ice beforeassaying. The protein concentration for each sample was measured byBradford assay. Proteasome chymotrypsin-like (CT-L) activity wasquantitated by monitoring the release of free AMC from the fluorogenicpeptide Suc-Leu-Leu-Val-Tyr-AMC (BostonBiochem).

As shown in FIG. 3, the CT-L activity was comparable between thestandard carfilzomib and Example conjugate 1 in the blood as well as inthe tissues of the adrenal gland, the heart and of the liver, in mice.

Example 42: Mean Carfilzomib Plasma Concentration—Time Profiles forExamples 13, 26 and 34, Each Administered IV

The PEG carfilzomib compounds were administered to mice (Balb/c, male,n=9 per dose group) as an intravenous (i.v.) bolus at 5 mg/kg(equivalent to carfilzomib, dose volume 1 mL/kg). Blood samples fromeach mouse were collected at the indicated time points (0.5, 1, 2, 4, 6,12, 16 and 24 hours post-dose) and 25 μL of the plasma samples wereextracted by protein precipitation with 125 μL of acetonitrilecontaining D10-CFZ as an internal standard and then centrifuged.Carfilzomib concentrations were measured in the supernatant by LC-MS/MSusing multiple reaction monitoring in the positive electrosprayionization mode. The lower limit of quantitation of the assay was 0.500ng/mL.

As shown in FIG. 4, the control standard carfilzomib (CFZ) cyclodextrinformulation (5 mg/mL) in this study resulted in a plasma concentrationdrop over a very short period of time. The 3K-PEG CFZ (Example 34) ispresent in the plasma for up to between 20 and about 25 hours postinitial administration of the PEG conjugate. Similarly, the 5K-PEG CFZ(Example 26) is present in the plasma, as measured in FIG. 5, at higherconcentrations for practically all of the time out to about 25 hours.Finally, the 20K-PEG (Example 13) is the curve above both the 3K and5K-PEG curves, while beginning between them, and revealing that this PEGconjugate releases carfilzomib into the plasma over the course of the 25hours measured, while providing carfilzomib at significantly higherplasma concentrations for that lengthy duration of time. Finally, asshown in FIG. 4, the highest most curve at the starting time point, aformulation comprising a combination of both the 3K-PEG and the 20K-PEGcarfilzomib compounds exhibited a plasma concentration both higher andlonger than the 3K-PEG individually and comparable to the highermolecular weight 20K-PEG carfilzomib compound alone.

Table 4 describes the results obtained when the representativecarfilzomib compound examples were compared with standard carfilzomiband all samples were dosed IV.

TABLE 4 pK Measurements Volume of Distribution Example C_(max) AUC₀₋₂₄CL/F (Vdss; No. (ng/mL) (ng · h/mL) T_(1/2) (h) (mL/kg/h) (mL/kg) CFZ-IV46.7 153 10.9 29000 245000 34 1470 2230 6.09 2230 2110 26 472 847 8.515770 12800 13 616 3510 3.68 1400 6900

The PEG carfilzomib formulations dosed in Examples 39-41, were generallyprepared as follows: A desired amount of the PEG-CFZ compound wasweighed out into a sterile glass container using an analytical balance.A diluent volume was calculated based on the weight of the material. Adiluent of 10 mM Acetate, pH 5.0, 9% sucrose was added to the glassvessel to a final volume that resulted in PEG-CFZ compounds'concentrations of 1 mg/ml, 5 mg/mL, 10 mg/mL or 20 mg/mL. Each PEG-CFZsample was stirred for an hour at room temperature to allow for fulldissolution of the material. Once the material was completely dissolvedin solution, a sample was taken to measure the pH, which wasconsistently found to be in the desired range of 4.9-5.1. Thus, nofurther pH adjustments were done. Sample osmolality measurements andendotoxin testing was performed for all samples and consistently foundto be within the acceptable range of 295-312 mOsm for osmolality and<1.0 EU/mL for endotoxin count. Immediately after dissolution sampleswere aseptically filled into 5 cc sterile glass vials, stoppered andcapped. The samples were frozen at −70° C. for a period of 1 day to 2.5weeks prior to transport and dosing in the examples 39-41 as describedherein below.

Example 43: Efficacy Study of Example 13 and CFZ-Captisol in HT-29 HumanColorectal Adenocarcinoma Xenograph Model in SCID Beige Mice (FIG. 5)

Procedure: Female Beige Severe Combined Immune Deficiency (SCID) mice(60 plus spares) were purchased from Harlan Laboratories (Livermore,Calif.) as 6- to 7-week-old mice. Following arrival, animals wereweighed using an electronic balance (Ohaus SCOUT® PRO, Parsippany,N.J.), given a clinical examination to ensure that the animals were ingood condition, and housed 5 per cage (prior to dosing). The animalswere maintained in a HEPA-filtered environment in a Micro-VENTfull-ventilation rodent housing system (Allentown Caging Equipment Co.,Allentown, N.J.) providing at least 10 room air changes per hour Animalroom controls were set to maintain temperature and relative humidity at20° C.±1° C. and 50%±20%, respectively. Housing rooms were on a 12:12light/dark cycle. Cages were autoclaved, and animals were bedded onSaniChip irradiated bedding 7990.BG (Harlan Teklad; Hayward, Calif.).Water was autoclaved and supplied ad libitum to each cage via waterbottles. Irradiated 2018 Teklad Global 16% Protein Rodent Diet ((HarlanTeklad) supplied ad libitum to each cage.

Compound Formulation:

Example 13 was prepared as generally described above. The carfilzomibcomparator compound was prepared as CFZ-captisol (at 1 mg/mL). A powdersample of example 13 was diluted to 30 mg/ml (Group 3) or 50 mg/ml(Group 3 beginning fourth dose) or 40 mg/ml (Group 4) in 10%ETOH/saline. Vehicle and CFZ-captisol were stored at 4° C. throughoutthe study. During the study, example 13 was inspected regularly forpotential changes in the quality of the suspension; none were observed

Cell Line:

NCI-HT29 (HT-29; ATCC® HTB-38™), a human colorectal adenocarcinomacancer cell (CA) line, was purchased from ATCC (Manassas, Va.).Following receipt at the MGI, cells were grown in-house for 7 passagesin RPMI 1640 and 10% fetal bovine serum, then used to generate frozenstocks. The cells were recovered from the frozen stocks and cultured asabove. Following growth, the cells were spun down and resuspended at aconcentration of 5E07 cells/mL in serum-free medium without additives,then combined 1:1 with Matrigel™ (Trevigen, Gaithersburg, Md.). At thetime of implant, cells corresponded to MGI Passage 7 (MGP7).

Implantation of Cells:

At approximately 3 weeks prior to the projected staging day, mice wereimplanted by subcutaneous (SC) injection into the lower left abdominalflank with 200 μL (5.0E06 cells) per mouse of the freshly preparedHT29:Matrigel mixture. All procedures were carried out in HEPA-filteredlaminar-flow hoods.

Study Design:

Study design and treatments of all groups are shown in Table I(Efficacy). When the tumors reached a mean volume of approximately 200mm³ per mouse, forty animals with established tumors and moderate bodyweights were randomized into 4 treatment groups (n=10 mice per group).Starting on Day 0, animals were administered by once-weekly (qw)injection with vehicle (Group 1) or twice-weekly D1D2 injection (i.e.,two adjacent days each week) with CFZ-captisol at 5 mpk (Group 2) orOP-59381 at 150 mg/kg (Groups 3). Starting with the fourth dose (i.e.,after three week), dosing of Group 3 animals was increased to 250 mg/kg.Group 4 was dosed once-weekly (qw) injection with or OP-59381 (Example13) at 200 mg/kg. All of these doses were administered as intravenous(IV) injections at dose volumes of 5 mL/kg. After the IV dosing for theseventh week (i.e., after Day 42 for Groups 1-4), the tumor treatmentefficacy appeared to slow or cease altogether.

TABLE 5 Days 0-48 Experiment Group Agent Dose (mg/kg) Frequency 1 1 (n =10) Vehicle — QW × 7 weeks 1 2 (n = 10) CFZ-captisol  5 QD × 2 (D1D2) ×7 weeks 1 3 (n = 10) Example 13 150/250 QW × 7 weeks 1 4 (n = 10)Example 13 200 QW × 7 weeks

As seen in FIG. 5, the tumors in the vehicle group (Group 1) grewlinearly over the IV dosing interval, with tumors increasing to ˜2,755%of initial size by Day 49. While Day 0 to Day 15 tumor sizes wereunchanged vs. vehicle control in all three groups, by Day 19 tumorgrowth was significantly attenuated in the 200 mpk Example 13 and the 5mpk CFZ-captisol treated animals. This significant attenuation continuedto Day 29 when all three experimental groups achieved significance thatcontinued to Day 40, demonstrating that the 3 doses were sufficient toprovide anti-tumor activity.

Example 44: Example 44 Reflects the Mouse Survival Data Resulting fromStudy of Example 41, which Data is Tabulated in Table 6 and GraphicallyIllustrated in FIG. 6

TABLE 6 % Day 0 Day 40 Change # of Tumor Tumor compared animals Vol Volto at day Group (mm³) (mm³) Vehicle 40 1 Vehicle 95.8 2471.6 0 3 2CFZ-Captisol 5mpk (D1D2) 95.6 1370.6 −44.5% 4 3 Example 13 - 150 mpk to250 mpk (QW) 95.5 998.5 −59.6% 9 4 Example 13 - 200 mpk (QW) 95.9 1443.0−41.6% 7

Examples 41 and 42 reveal the efficacy of carfilzomib representativecompound Example 13 in a mouse xenograft model of human colorectaladenocarcinoma cancer cell. Tumors in the vehicle group grew linearlyduring the study. Once-weekly intravenous dosing with compound Example13 (200 mpk, or 150 rising to 250 mpk after 3 week) or with CFZ-captisol(5 mpk) provided significant attenuation of tumor growth (compared tovehicle control) within 19 days of the first dose administration. Inaddition, intravenous dosing with the both formulations was associatedwith significant attenuation of weight gain.

Example 45: Efficacy Study of Example 13 and CFZ-Captisol in HT-29Colorectal Adenocarcinoma Xenograph Model in SCID Beige Mice (FIG. 7)Procedure:

Female Beige Severe Combined Immune Deficiency (SCID) mice (60 plusspares) were purchased from Harlan Laboratories (Livermore, Calif.) as6- to 7-week-old mice. Following arrival, animals were weighed using anelectronic balance (Ohaus SCOUT® PRO, Parsippany, N.J.), given aclinical examination to ensure that the animals were in good condition,and housed 5 per cage (prior to dosing). The animals were maintained ina HEPA-filtered environment in a Micro-VENT full-ventilation rodenthousing system (Allentown Caging Equipment Co., Allentown, N.J.)providing at least 10 room air changes per hour Animal room controlswere set to maintain temperature and relative humidity at 20° C.±1° C.and 50%±20%, respectively. Housing rooms were on a 12:12 light/darkcycle. Cages were autoclaved, and animals were bedded on SaniChipirradiated bedding 7990.BG (Harlan Teklad; Hayward, Calif.). Water wasautoclaved and supplied ad libitum to each cage via water bottles.Irradiated 2018 Teklad Global 16% Protein Rodent Diet ((Harlan Teklad)supplied ad libitum to each cage.

Compound Formulation:

Example 13 was formulated as described herein to the desiredconcentration. Carfilzomib was provided as CFZ-captisol (at 1 mg/mL). apowder sample of Example 13 was diluted to 30 mg/ml (Group 3) or 50mg/ml (Group 4) in 10% ETOH/saline. Vehicle and CFZ-captisol were storedat 4° C. throughout the study. During the study, example 13 preparationswere inspected regularly for potential changes in the quality of thesuspension; none were observed.

Cell Line:

NCI-HT29 (HT-29; ATCC® HTB-38™), a human colorectal adenocarcinomacancer cell (CA) line, was purchased from ATCC (Manassas, Va.).Following receipt at the MGI, cells were grown in-house for 7 passagesin RPMI 1640 and 10% fetal bovine serum, then used to generate frozenstocks. The cells were recovered from the frozen stocks and cultured asabove. Following growth, the cells were spun down and resuspended at aconcentration of 5E07 cells/mL in serum-free medium without additives,then combined 1:1 with Matrigel™ (Trevigen, Gaithersburg, Md.). At thetime of implant, cells corresponded to MGI Passage 7 (MGP7).

Implantation of Cells:

At approximately 3 weeks prior to the projected staging day, mice wereimplanted by subcutaneous (SC) injection into the lower left abdominalflank with 200 μL (5.0E06 cells) per mouse of the freshly preparedHT29:Matrigel mixture. All procedures were carried out in HEPA-filteredlaminar-flow hoods.

Study Design:

Study design and treatments of all groups are shown in Table I(efficacy). When the tumors reached a mean volume of approximately 200mm³ per mouse, forty animals with established tumors and moderate bodyweights were randomized into 4 treatment groups (n=10 mice per group).Starting on Day 0, animals were administered by once-weekly (qw)injection with vehicle (Group 1) or twice-weekly D1D2 injection (i.e.,two adjacent days each week) with CFZ-captisol at 5 mpk (Group 2) orexample 13 at 150 mg/kg or 250 mg/kg (Groups 3 and 4, respectively). Allof these doses were administered as intravenous (IV) injections at dosevolumes of 5 mL/kg. After the sixth week's dose administration(s) (i.e.,after Day 35), IV dosing for efficacy ceased.

As seen in FIG. 7, tumors in the vehicle group (Group 1) grew linearlyover the IV dosing interval, with tumors increasing to ˜2100% of initialsize by Day 41. In comparison, tumor growth was significantly attenuatedin all of the experimental groups: Day-41 tumor sizes were 83.6%, 79.6%,and 61.8% of control for Groups 2, 3, and 4 respectively. Thisattenuation achieved significance starting at the earliest time points(9 or 12 days), demonstrating that two doses were sufficient to provideanti-tumor activity. Tumor growth resumed following the end of IV dosing(after Day 35).

TABLE 7 Days 0-48 Group Agent Dose Frequency 1 (n = 10) Vehicle — QW × 6weeks 2 (n = 10) CFZ-captisol  5 mg/kg QD × 2 (D1D2) × 6 weeks 3 (n =10) Example 13 150 mg/kg QD × 2 (D1D2) × 6 weeks 4 (n = 10) Example 13250 mg/kg QW × 6 weeks

Example 46: Proteosome Inhibition of Exemplary Compounds in Cells (FIGS.9-14) Cell Line:

MOLT-4 human acute lymphoblastic leukemia T lymphoblasts were grown forat least 6 passages in growth media (RPMI-1640 basal media supplementedwith 10% FBS and 1×L-glutamine). Suspension cells were plated into a96-well plate at a rate of 1-2e6 cell/mL (50 uL, ˜60,000 cells/well) induplicate.

Treatment:

Preparations of carfilzomib and pegylated carfilzomib compound examples5, 35, 36, 37, and 38 were dissolved in DMSO at a concentration of 10mM. Serial dilutions were performed in DMSO to produce concentrationscovering 7 logs, and then each serial dilution was further diluted 40×in growth media. Dilutions were then added in equal volume to wellscontaining cells, further diluting compound concentrations by two-fold.Cells were incubated at 37° C. (5% CO₂) for 1 hour, then spun at 1500rpm for 5 mins at RT. Media was removed and the cells were washed withPBS 3 times. After the last wash, supernatant was removed and the cellpellets were frozen on dry ice.

Analysis:

Cell pellets were thawed and then lysed by resuspending in 50 uL lysisbuffer on ice (20 mM Tris-HCl, 5 mM EDTA, pH 8). The preparation wascentrifuged at 1500 rpm for 5 mins and then utilized directly forAMC-LLVY fluorescent assay. Measurements were taken every 2 minutes for70 minutes to generate a kinetic curve, and IC₅₀ values were calculatedusing the 5-15 minute slope (RFU/min, initial velocity).

As shown in FIG. 10, representative exemplary compound 5 exhibited acellular proteasome chymotrypsin-like (CT-L) IC₅₀ inhibitory potency ofabout 16.25 nM. By comparison, the control sample of carfilzomibexhibited an IC₅₀ activity of about 9.2 nM, a potency similar to that ofexample 5. Similarly, compound example No. 35 (FIG. 11) exhibited acellular proteasome chymotrypsin-like (CT-L) IC₅₀ inhibitory potency ofabout 9.0 nM, compound example no. 36 (FIG. 12) exhibited a cellularproteasome chymotrypsin-like (CT-L) IC₅₀ inhibitory potency of about21.8 nM, compound example no. 37 (FIG. 13) exhibited a cellularproteasome chymotrypsin-like (CT-L) IC₅₀ inhibitory potency of about22.8 nM and compound example no. 38 (FIG. 14) exhibited a cellularproteasome chymotrypsin-like (CT-L) IC₅₀ inhibitory potency of about21.8 nM. Some representative compounds of the present invention exhibitCT-L inhibitory activity as a pegylated carfilzomib compound.

Example 47: Proteasome Inhibition of PEG-Carfilzomib Conjugates Vs.Carfilzomib

PEG carfilzomib compound examples 35 and 36 were administered to mice(Balb/c, female, n=3 per dose group) as a subcutaneous bolus at thespecified dose (dose volume 5 mL/kg) in an aqueous solution containing10 mmol/L sodium acetate (pH 5.0) and 9% sucrose for administration (20mg/kg). At selected time points after drug administration, blood sampleswere collected using tubes containing heparin. Samples are immediatelyplaced on ice and spun at maximum speed for 2 minutes in amicrocentrifuge at room temperature (RT). Cell pellets are stored on wetice. Whole blood cell pellets are resuspended in 1 ml of phosphatebuffered saline (PBS) and centrifuged at max speed at 4° C. Supernatantsare removed and the pellets are washed a second time with PBS. Samplesare resuspended in 2 volumes of lysis buffer (20 mM Tris, pH 8.0, 5 mMEDTA) then frozen and stored at −80° C. until analysis.

Sample Processing:

All samples were thawed on ice. All single cell pellets in lysis buffer(whole blood) were briefly vortexed then spun at 14,000 rpm in amicrocentrifuge at 4° C. for 15 minutes. Supernatant was transferred ata ratio of 100 μL to 25 μL of 50% glycerol in a sample plate for a finalconcentration of 10% glycerol. These samples were then ready forassaying, or they may be frozen at −80° C. Samples frozen at thislysate/10% glycerol stage should be thawed on ice before assaying. Theprotein concentration for each sample was measured by Bradford assay.Proteasome chymotrypsin-like (CT-L) activity was quantitated bymonitoring the release of free AMC from the fluorogenic peptideSuc-Leu-Leu-Val-Tyr-AMC (BostonBiochem).

FIG. 15 depicts the in-vivo pharmacodynamic (PD) activity ofrepresentative compound examples 35 and 36 of the present invention. Asshown in FIG. 15, each of examples 35 and 36 exhibited in vivo nearlycomplete CT-L inhibition for over 48 h after administration in a mouse.Even over the duration of 96 hrs out, the compounds continued to exhibitCT-L inhibitory activity of about 50% or more in the mouse.

Methods of Use

The biological effects of proteasome inhibition are useful anddesirable. Proteasome inhibition has been suggested as a preventionand/or treatment of a multitude of diseases including, but not limitedto, proliferative diseases, neurotoxic/degenerative diseases,Alzheimer's, ischemic conditions, inflammation, auto-immune diseases,HIV, cancers, organ graft rejection, septic shock, inhibition of antigenpresentation, decreasing viral gene expression, parasitic infections,conditions associated with acidosis, macular degeneration, pulmonaryconditions, muscle wasting diseases, fibrotic diseases, bone and hairgrowth diseases. Therefore, pharmaceutical formulations comprising thePEG carfilzomib compounds of the invention in therapeutically effectivedosage amounts provide a means of administering a drug to a patient andtreating these conditions.

At the cellular level, the accumulation of polyubiquitinated proteins,cell morphological changes, and apoptosis have been reported upontreatment of cells with various proteasome inhibitors. Proteasomeinhibition has also been disclosed, and clinically and commerciallyproven as a useful antitumor therapeutic strategy. To this end, thecompounds and compositions including the compounds of the presentinvention are useful for treating cancer, including without limitation,newly diagnosed and/or relapsed and refractory multiple myeloma.

Both in vitro and in vivo models have shown that malignant cells, ingeneral, are susceptible to proteasome inhibition. In fact, proteasomeinhibition has already been validated as a therapeutic strategy for thetreatment of multiple myeloma. This could be due, in part, to the highlyproliferative malignant cell's dependency on the proteasome system torapidly remove proteins (Rolfe et al., J. Mol. Med. (1997) 75:5-17;Adams, Nature (2004) 4: 349-360). Provided herein is a method oftreating cancer comprising administering to a patient in need of suchtreatment a therapeutically effective amount of a pegylated carfilzomibcompound of formulas I and II, or any specifically exemplified PEGcarfilzomib compound, as provided or described herein.

As used herein, the term “cancer” includes, but is not limited to, bloodborne cancers and solid tumors. Cancer may afflict components of blood,bone, organs, skin tissue and the vascular system, including, but notlimited to, cancers of the bladder, blood, bone, brain, breast, cervix,chest, colon, endrometrium, esophagus, eye, head, kidney, liver, lung,lymph nodes, mouth, neck, ovaries, pancreas, prostate, rectum, renal,skin, stomach, testis, throat, and uterus. Specific cancers include, butare not limited to, leukemia (acute lymphocytic leukemia (ALL), acutemyelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronicmyelogenous leukemia (CML), hairy cell leukemia), mature B cellneoplasms (small lymphocytic lymphoma), B cell prolymphocytic leukemia,lymphoplasmacytic lymphoma (such as Waldenström's macroglobulinemia),splenic marginal zone lymphoma, plasma cell myeloma, plasmacytoma,monoclonal immunoglobulin deposition diseases, heavy chain diseases,extranodal marginal zone B cell lymphoma (MALT lymphoma), nodal marginalzone B cell lymphoma (NMZL), follicular lymphoma, mantle cell lymphoma,diffuse B cell lymphoma, mediastinal (thymic) large B cell lymphoma,intravascular large B cell lymphoma, primary effusion lymphoma andBurkitt lymphoma/leukemia), mature T cell and natural killer (NK) cellneoplasms (T cell prolymphocytic leukemia, T cell large granularlymphocytic leukemia, aggressive NK cell leukemia, adult T cellleukemia/lymphoma, extranodal NK/T cell lymphoma, enteropathy-type Tcell lymphoma, hepatosplenic T cell lymphoma, blastic NK cell lymphoma,mycosis fungoides (Sezary syndrome), primary cutaneous anaplastic largecell lymphoma, lymphomatoid papulosis, angioimmunoblastic T celllymphoma, unspecified peripheral T cell lymphoma and anaplastic largecell lymphoma), Hodgkin lymphoma (nodular sclerosis, mixed celluarity,lymphocyte-rich, lymphocyte depleted or not depleted, nodularlymphocyte-predominant), non-hodgkin's lymphoma, myeloma (multiplemyeloma, indolent myeloma, smoldering myeloma), chronicmyeloproliferative disease, myelodysplastic/myeloproliferative disease,myelodysplastic syndromes, immunodeficiency-associatedlymphoproliferative disorders, histiocytic and dendritic cell neoplasms,mastocytosis, chondrosarcoma, Ewing sarcoma, fibrosarcoma, malignantgiant cell tumor, myeloma bone disease, osteosarcoma, breast cancer(hormone dependent, hormone independent), gynecological cancers(cervical, endometrial, fallopian tube, gestational trophoblasticdisease, ovarian, peritoneal, uterine, vaginal and vulvar), basal cellcarcinoma (BCC), squamous cell carcinoma (SCC), malignant melanoma,dermatofibrosarcoma protuberans, Merkel cell carcinoma, Kaposi'ssarcoma, astrocytoma, pilocytic astrocytoma, dysembryoplasticneuroepithelial tumor, oligodendrogliomas, ependymoma, glioblastomamultiforme, mixed gliomas, oligoastrocytomas, medulloblastoma,retinoblastoma, neuroblastoma, germinoma, teratoma, malignantmesothelioma (peritoneal mesothelioma, pericardial mesothelioma, pleuralmesothelioma), gastro-entero-pancreatic or gastroenteropancreaticneuroendocrine tumor (GEP-NET), carcinoid, pancreatic endocrine tumor(PET), colorectal adenocarcinoma, colorectal carcinoma, aggressiveneuroendocrine tumor, leiomyosarcomamucinous adenocarcinoma, Signet Ringcell adenocarcinoma, hepatocellular carcinoma, cholangiocarcinoma,hepatoblastoma, hemangioma, hepatic adenoma, focal nodular hyperplasia(nodular regenerative hyperplasia, hamartoma), non-small cell lungcarcinoma (NSCLC) (squamous cell lung carcinoma, adenocarcinoma, largecell lung carcinoma), small cell lung carcinoma, thyroid carcinoma,prostate cancer (hormone refractory, androgen independent, androgendependent, hormone-insensitive), and soft tissue sarcomas (fibrosarcoma,malignant fibrous hystiocytoma, dermatofibrosarcoma, liposarcoma,rhabdomyosarcoma leiomyosarcoma, hemangiosarcoma, synovial sarcoma,malignant peripheral nerve sheath tumor/neurofibrosarcoma, extraskeletalosteosarcoma).

In one aspect, the invention provides a pegylated carfilzomib compoundas provided herein, or a pharmaceutical composition comprising the same,can be administered to treat multiple myeloma in a patient. For example,multiple myeloma can include either or both newly diagnosed or relapsedand/or refractory multiple myeloma.

Many tumors of the haematopoietic and lymphoid tissues are characterizedby an increase in cell proliferation, or a particular type of cell. Thechronic myeloproliferative diseases (CMPDs) are clonal haematopoieticstem cell disorders characterized by proliferation in the bone marrow ofone or more of the myeloid lineages, resulting in increased numbers ofgranulocytes, red blood cells and/or platelets in the peripheral blood.As such, use of a proteasome inhibitor for the treatment of suchdiseases is attractive and being examined (Cilloni et al., Haematologica(2007) 92: 1124-1229). CMPD can include chronic myelogenous leukemia,chronic neutrophilic leukemia, chronic eosinophilic leukemia,polycythaemia vera, chronic idiopathic myelofibrosis, essentialthrombocythaemia and unclassifiable chronic myeloproliferative disease.Provided herein is a method of treating CMPD comprising administering toa patient in need of such treatment an effective amount of theproteasome inhibitor compound disclosed herein.

Myelodisplastic/myeloproliferative diseases, such as chronicmyelomonocytic leukemia, atypical chronic myeloid leukemia, juvenilemyelomonocytic leukemia and unclassifiablemyelodysplastic/myeloproliferative disease, are characterized byhypercellularity of the bone marrow due to proliferation in one or moreof the myeloid lineages. Inhibiting the proteasome with a compositiondescribed herein, can serve to treat thesemyelodisplatic/myeloproliferative diseases by providing a patient inneed of such treatment an effective amount of the composition.

Myelodysplastic syndromes (MDS) refer to a group of hematopoietic stemcell disorders characterized by dysplasia and ineffective haematopoiesisin one or more of the major myeloid cell lines. Targeting NF-kB with aproteasome inhibitor in these hematologic malignancies inducesapoptosis, thereby killing the malignant cell (Braun et al. Cell Deathand Differentiation (2006) 13:748-758). Further provided herein is amethod to treat MDS comprising administering to a patient in need ofsuch treatment an effective amount of a compound provided herein. MDSincludes refractory anemia, refractory anemia with ringed sideroblasts,refractory cytopenia with multilineage dysplasia, refractory anemia withexcess blasts, unclassifiable myelodysplastic syndrome andmyelodysplastic syndrome associated with isolated del (5q) chromosomeabnormality.

Mastocytosis is a proliferation of mast cells and their subsequentaccumulation in one or more organ systems. Mastocytosis includes, but isnot limited to, cutaneous mastocytosis, indolent systemic mastocytosis(ISM), systemic mastocytosis with associated clonal haematologicalnon-mast-cell-lineage disease (SM-AHNMD), aggressive systemicmastocytosis (ASM), mast cell leukemia (MCL), mast cell sarcoma (MCS)and extracutaneous mastocytoma. Further provided herein is a method totreat mastocytosis comprising administering an effect amount of thecompound disclosed herein to a patient diagnosed with mastocytosis.

Additional embodiments include methods for affecting theproteasome-dependent regulation of oncoproteins and methods of treatingor inhibiting cancer growth, each method including exposing a cell (invivo, e.g., in a patient, or in vitro) to a composition disclosedherein. HPV-16 and HPV-18-derived E6 proteins stimulate ATP- andubiquitin-dependent conjugation and degradation of p53 in crudereticulocyte lysates. The recessive oncogene p53 has been shown toaccumulate at the nonpermissive temperature in a cell line with amutated thermolabile E1. Elevated levels of p53 may lead to apoptosis.Examples of proto-oncoproteins degraded by the ubiquitin system includec-Mos, c-Fos, and c-Jun. One embodiment is a method for treatingp53-related apoptosis, including administering to a patient an effectiveamount of a composition disclosed herein.

It has also been demonstrated that inhibitors that bind to the 20Sproteasome stimulate bone formation in bone organ cultures. Furthermore,when such inhibitors have been administered systemically to mice,certain proteasome inhibitors increased bone volume and bone formationrates over 70% (Garrett, I. R. et al., J. Clin. Invest. (2003) 111:1771-1782), therefore suggesting that the ubiquitin-proteasome machineryregulates osteoblast differentiation and bone formation. Therefore, thedisclosed compositions may be useful in the treatment and/or preventionof diseases associated with bone loss, such as osteoporosis.

Bone tissue is an excellent source for factors which have the capacityfor stimulating bone cells. Thus, extracts of bovine bone tissue containnot only structural proteins which are responsible for maintaining thestructural integrity of bone, but also biologically active bone growthfactors which can stimulate bone cells to proliferate. Among theselatter factors are a recently described family of proteins called bonemorphogenetic proteins (BMPs). All of these growth factors have effectson other types of cells, as well as on bone cells, including Hardy, M.H., et al., Trans Genet (1992) 8:55-61 describes evidence that bonemorphogenetic proteins (BMPs), are differentially expressed in hairfollicles during development. Harris, S. E., et al., J Bone Miner Res(1994) 9:855-863 describes the effects of TGF-β on expression of BMP-2and other substances in bone cells. BMP-2 expression in mature folliclesalso occurs during maturation and after the period of cell proliferation(Hardy, et al. (1992, supra). Thus, compounds provided herein may alsobe useful for hair follicle growth stimulation.

Finally, the disclosed compositions are also useful as diagnostic agents(e.g., in diagnostic kits or for use in clinical laboratories) forscreening for proteins (e.g., enzymes, transcription factors) processedby Ntn hydrolases, including the proteasome. The disclosed compositionsare also useful as research reagents for specifically binding the X/MB1subunit or α-chain and inhibiting the proteolytic activities associatedwith it. For example, the activity of (and specific inhibitors of) othersubunits of the proteasome can be determined.

Most cellular proteins are subject to proteolytic processing duringmaturation or activation. Enzyme inhibitors disclosed herein can be usedto determine whether a cellular, developmental, or physiological processor output is regulated by the proteolytic activity of a particular Ntnhydrolase. One such method includes obtaining an organism, an intactcell preparation, or a cell extract; exposing the organism, cellpreparation, or cell extract to a composition disclosed herein; exposingthe compound-exposed organism, cell preparation, or cell extract to asignal, and monitoring the process or output. The high selectivity ofthe compounds disclosed herein permits rapid and accurate elimination orimplication of the Ntn (for example, the 20S proteasome) in a givencellular, developmental, or physiological process.

Pharmaceutical Compositions

The present invention further provides pharmaceutical compositions toadminister the treatment for cancer patients. The compositions compriseone, and in some embodiments more than one, PEG carfilzomib compound ofFormulas I or II, and sub-formulas thereof, as described herein. Onetype of pharmaceutical composition the present invention provides is aparenterally administered pharmaceutical composition. Parenterallyadministrable compositions suitable for infusion, injection orsub-cutaneous administration can include sterile aqueous solutions(where water soluble) or dispersions and/or sterile powders for theextemporaneous preparation of sterile solutions or dispersions foreither infusion or injection. For intravenous administration, such as byinfusion, suitable carriers include sterile water for injection, sterilebuffers, such as citrate buffer, bacteriostatic water, and Cremophor EL™(BASF, Parsippany, N.J.). In all cases, the composition, particularlyfor human use, treatment and consumption, must be sterile and should befluid to the extent that it is easy to add to or pull up into a syringeor infusion bag. The composition should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants. Prevention of theaction of microorganisms can be achieved by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal, and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, and sodium chloride in the composition.Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent that delays absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompounds of the invention, PEG carfilzomib compounds, in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thecarfilzomib into a sterile vehicle, which contains a basic dispersionmedium and the required other ingredients from those enumerated above.In the case of sterile powders for the preparation of sterile injectablesolutions, a suitable method of preparation is freeze-drying(lyophilization), which provides a powder form of the carfilzomib plusany additional desired ingredient from a previously sterile-filteredsolution thereof.

Systemic administration of a therapeutic compound of the invention asdescribed herein can also be by transmucosal or transdermal means. Fortransmucosal or transdermal administration, penetrants appropriate tothe barrier to be permeated are used in the formulation. Such penetrantsare generally known in the art, and include, for example, fortransmucosal administration, detergents, bile salts, and fusidic acidderivatives. Transmucosal administration can be accomplished through theuse of nasal sprays or suppositories. For transdermal administration,the active compounds of the invention are formulated into ointments,salves, gels, or creams as generally known in the art.

In one embodiment, the therapeutic PEG carfilzomib compounds of theinvention are prepared with carriers that will protect the therapeuticcompounds against rapid elimination from the body, such as a controlledrelease formulations. Other examples include, without limitation,implants and microencapsulated delivery systems. Biodegradable,biocompatible polymers can be used, such as ethylene vinyl acetate,polyanhydrides, poly glycolic acid, collagen, polyorthoesters, andpolylactic acid. Such formulations can be prepared using standardtechniques, or obtained commercially, e.g., from Alza Corporation andNova Pharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to selected cells with monoclonal antibodies to cellularantigens) can also be used as pharmaceutically acceptable carriers.These can be prepared according to methods known to those skilled in theart, for example, as described in U.S. Pat. No. 4,522,811.

The pharmaceutical compositions provided by the present invention may beadministered at once, or may be divided into a number of smaller dosesto be administered at intervals of time. It is understood that theprecise dosage and duration of treatment is a function of the diseasebeing treated and may be determined empirically using known testingprotocols or by extrapolation from in vivo or in vitro test data. It isto be noted that concentrations and dosage values may also vary with theseverity of the condition to be alleviated. It is to be furtherunderstood that for any particular patient, specific dosage regimensshould be adjusted over time according to the individual need and theprofessional judgment of the person administering or supervising theadministration of the compositions, and that the concentration rangesset forth herein are exemplary only and are not intended to limit thescope or practice of the claimed compositions.

Dosage forms or compositions containing a PEG-carfilzomib compound ofthe invention as described herein in the range of 0.005% to 100% withthe balance made up from non-toxic carrier may be prepared. Methods forpreparation of these compositions are known to those skilled in the art.The contemplated pharmaceutical compositions of the invention maycontain 0.001%-100% of the PEG carfilzomib compound provided herewith,in one embodiment 0.1-95%, and in another embodiment 75-85%. Thepharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration. In someembodiments, the PEG carfilzomib compounds provided herein may beformulated as described in U.S. Pat. No. 9,309,283.

Administration

Compositions prepared as described herein can be administered in variousforms, depending on the disorder to be treated and the age, condition,and body weight of the patient, as is well known in the art. Forexample, where the compositions are to be administered orally, they maybe formulated as tablets, capsules, granules, powders, or syrups; or forparenteral administration, they may be formulated as injections(intravenous, intramuscular, or subcutaneous), drop infusionpreparations, or suppositories. For application by the ophthalmic mucousmembrane route, they may be formulated as eye drops or eye ointments.These formulations can be prepared by conventional means in conjunctionwith the methods described herein, and, if desired, the activeingredient may be mixed with any conventional additive or excipient,such as a binder, a disintegrating agent, a lubricant, a corrigent, asolubilizing agent, a suspension aid, an emulsifying agent, or a coatingagent. Although the dosage will vary depending on the symptoms, age andbody weight of the patient, the nature and severity of the disorder tobe treated or prevented, the route of administration and the form of thedrug, in general, a daily dosage of from 0.01 to 2000 mg of the compoundis recommended for an adult human patient, and this may be administeredin a single dose or in divided doses. The amount of active ingredientwhich can be combined with a carrier material to produce a single dosageform will generally be that amount of the compound which produces atherapeutic effect. More information on the dosage amounts for compoundsof the invention is provided herein below. In general, compositionsintended for parenteral use (e.g., intravenous, subcutaneous injection)include a solubilizing agent. The solubilizing agent may be asubstituted cyclodextrin.

The precise time of administration and/or amount of the composition thatwill yield the most effective results in terms of efficacy of treatmentin a given patient will depend upon the activity, pharmacokinetics, andbioavailability of the particular PEG carfilzomib compound,physiological condition of the patient (including age, sex, disease typeand stage, general physical condition, responsiveness to a given dosage,and type of medication), route of administration, and the like. However,the above guidelines can be used as the basis for fine-tuning thetreatment, e.g., determining the optimum time and/or amount ofadministration, which will require no more than routine experimentationconsisting of monitoring the patient and adjusting the dosage and/ortiming.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose ligands, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition, or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial. Each carrier must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation and notinjurious to the patient. Some examples of materials which can serve aspharmaceutically acceptable carriers include: (1) sugars, such aslactose, glucose, and sucrose; (2) starches, such as corn starch, potatostarch, and substituted or unsubstituted β-cyclodextrin; (3) cellulose,and its derivatives, such as sodium carboxymethyl cellulose, ethylcellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6)gelatin; (7) talc; (8) excipients, such as cocoa butter and suppositorywaxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil,sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such aspropylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol,and polyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20)phosphate buffer solutions; and (21) other non-toxic compatiblesubstances employed in pharmaceutical formulations. In certainembodiments, pharmaceutical compositions provided herein arenon-pyrogenic, i.e., do not induce significant temperature elevationswhen administered to a patient.

The term “pharmaceutically acceptable salt” refers to the relativelynon-toxic, inorganic and organic acid addition salts or basic salts ofthe compound(s) of the invention. These salts can be prepared in situduring the final isolation and purification of the compound(s), or byseparately reacting a purified PEG-carfilzomib compound in its free baseform with a suitable organic or inorganic acid, and isolating the saltthus formed. Representative acid addition salts include thehydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate,acetate, valerate, oleate, palmitate, stearate, laurate, benzoate,lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate,tartrate, naphthylate, mesylate, glucoheptonate, lactobionate,laurylsulphonate salts, and amino acid salts, and the like. (See, forexample, Berge et al. (1977) “Pharmaceutical Salts”, J. Pharm. Sci. 66:1-19.)

In some embodiments, the PEG carfilzomib compounds provided herein maycontain one or more acidic functional groups and, thus, are capable offorming pharmaceutically acceptable salts with pharmaceuticallyacceptable bases. The term “pharmaceutically acceptable salts” in theseinstances refers to the relatively non-toxic inorganic and organic baseaddition salts of the compound. These salts can likewise be prepared insitu during the final isolation and purification of the inhibitor(s), orby separately reacting the compound in its free acid form with asuitable base, such as the hydroxide, carbonate, or bicarbonate of apharmaceutically acceptable metal cation, with ammonia, or with apharmaceutically acceptable organic primary, secondary, or tertiaryamine Representative basic salts include, without limitation, alkali oralkaline earth salts such as lithium, sodium, potassium, calcium,magnesium, and aluminum salts, and the like. Representative organicamines useful for the formation of base addition salts includeethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine,piperazine, and the like (see, for example, Berge et al., supra).

Wetting agents, emulsifiers, and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring, and perfuming agents,preservatives and antioxidants can also be present in the compositions.

Examples of pharmaceutically acceptable antioxidants include: (1) watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like;(2) oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Formulations suitable for oral administration may be in the form ofcapsules, cachets, pills, tablets, lozenges (using a flavored basis,usually sucrose and acacia or tragacanth), powders, granules, or as asolution or a suspension in an aqueous or non-aqueous liquid, or as anoil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup,or as pastilles (using an inert matrix, such as gelatin and glycerin, orsucrose and acacia) and/or as mouthwashes, and the like, each containinga predetermined amount of compound of the invention as an activeingredient. A composition may also be administered as a bolus,electuary, or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), the active ingredient(compound of the invention) is mixed with one or more pharmaceuticallyacceptable carriers, such as sodium citrate or dicalcium phosphate,and/or any of the following: (1) fillers or extenders, such as starches,cyclodextrins, lactose, sucrose, glucose, mannitol, and/or silicic acid;(2) binders, such as, for example, carboxymethylcellulose, alginates,gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3) humectants,such as glycerol; (4) disintegrating agents, such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and sodium carbonate; (5) solution retarding agents, such as paraffin;(6) absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, acetyl alcohol and glycerolmonostearate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such a talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets, and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may also be employed as fillers in softand hard-filled gelatin capsules using such excipients as lactose ormilk sugars, as well as high molecular weight polyethylene glycols, andthe like.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered inhibitor(s)moistened with an inert liquid diluent.

Tablets, and other solid dosage forms, such as dragees, capsules, pills,and granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes, and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved in sterile water, or some other sterile injectable mediumimmediately before use. These compositions may also optionally containopacifying agents and may be of a composition that they release theactive ingredient(s) only, or preferentially, in a certain portion ofthe gastrointestinal tract, optionally, in a delayed manner. Examples ofembedding compositions which can be used include polymeric substancesand waxes. The active ingredient can also be in micro-encapsulated form,if appropriate, with one or more of the above-described excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredient, the liquid dosageforms may contain inert diluents commonly used in the art, such as, forexample, water or other solvents, solubilizing agents, and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, oils (in particular, cottonseed, groundnut, corn, germ, olive,castor, and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols, and fatty acid esters of sorbitan, and mixturesthereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active inhibitor(s) may containsuspending agents as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

Formulations for rectal or vaginal administration may be presented as asuppository, which may be prepared by mixing one or more inhibitor(s)with one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or a salicylate, which is solid at room temperature, butliquid at body temperature and, therefore, will melt in the rectum orvaginal cavity and release the active agent.

Formulations which are suitable for vaginal administration also includepessaries, tampons, creams, gels, pastes, foams, or spray formulationscontaining such carriers as are known in the art to be appropriate.

Dosage forms for the topical or transdermal administration of a selectedcompound of the invention include powders, sprays, ointments, pastes,creams, lotions, gels, solutions, patches, and inhalants. The compoundmay be mixed under sterile conditions with a pharmaceutically acceptablecarrier, and with any preservatives, buffers, or propellants which maybe required.

The ointments, pastes, creams, and gels may contain, in addition to thecompound(s) of the invention, excipients, such as animal and vegetablefats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc, andzinc oxide, or mixtures thereof. Powders and sprays can contain, inaddition to compound(s) of the invention, excipients such as lactose,talc, silicic acid, aluminum hydroxide, calcium silicates, and polyamidepowder, or mixtures of these substances. Sprays can additionally containcustomary propellants, such as chlorofluorohydrocarbons and volatileunsubstituted hydrocarbons, such as butane and propane.

A PEG carfilzomib compound of the invention can be administered byaerosol. This is accomplished by preparing an aqueous aerosol, liposomalpreparation, or solid particles containing the composition. Anon-aqueous (e.g., fluorocarbon propellant) suspension could be used. Insome embodiments, sonic nebulizers are preferred because they minimizeexposing the agent to shear, which can result in degradation of thecompound. Ordinarily, an aqueous aerosol is made by formulating anaqueous solution or suspension of the agent together with conventionalpharmaceutically acceptable carriers and stabilizers. The carriers andstabilizers vary with the requirements of the particular composition,but typically include nonionic surfactants (Tweens, Pluronics, sorbitanesters, lecithin, Cremophors), pharmaceutically acceptable co-solventssuch as polyethylene glycol, innocuous proteins like serum albumin,sorbitan esters, oleic acid, lecithin, amino acids such as glycine,buffers, salts, sugars, or sugar alcohols. Aerosols generally areprepared from isotonic solutions.

Transdermal patches have the added advantage of providing controlleddelivery of a compound of the invention to the body. Such dosage formscan be made by dissolving or dispersing the compound in the propermedium. Absorption enhancers can also be used to increase the flux ofthe compound(s) across the skin. The rate of such flux can be controlledby either providing a rate controlling membrane or dispersing thecompound(s) in a polymer matrix or gel.

Pharmaceutical compositions suitable for parenteral administrationcomprise one or more compound of the invention in combination with oneor more pharmaceutically acceptable sterile aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions provided herein includewater for injection, such as by sub-cutaneous administration (e.g.,sterile water for injection), ethanol, polyols (such as glycerol,propylene glycol, polyethylene glycol, and the like), buffer (such ascitrate buffer), and suitable mixtures thereof, vegetable oils, such asolive oil, and injectable organic esters, such as ethyl oleate. Properfluidity can be maintained, for example, by the use of coatingmaterials, such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

Pharmaceutical compositions typically include a pharmaceuticallyacceptable carrier. As used herein the language “pharmaceuticallyacceptable carrier” includes a buffer, sterile water for injection,solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. In some embodiments, apharmaceutically acceptable carrier is an acid-base buffer system, suchas a citrate buffer, to maintain a stable pH for the resulting solution.In some embodiments, a pharmaceutically acceptable carrier is sterilewater for injection. In some embodiments, a pharmaceutically acceptablecarrier comprises citric acid.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents, and dispersing agents. Prevention ofthe action of microorganisms may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include tonicity-adjusting agents, such as sugars and thelike into the compositions. In addition, prolonged absorption of theinjectable pharmaceutical form may be brought about by the inclusion ofagents which delay absorption such as aluminum monostearate and gelatin.In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. For example, delayed absorption of a parenterallyadministered drug form is accomplished by dissolving or suspending thedrug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe compound(s) of the invention in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio, typically weightratio, of carfilzsomib to polymer, and the nature of the particularpolymer employed, the rate of carfilzomb release can be controlled.Examples of other biodegradable polymers include poly(orthoesters) andpoly(anhydrides). Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissue.

The preparations of agents may be given orally, parenterally, topically,or rectally. They are, of course, given by forms suitable for eachadministration route. For example, they are administered in tablets orcapsule form, by injection, inhalation, eye lotion, ointment,suppository, infusion; topically by lotion or ointment; and rectally bysuppositories. In some embodiments, administration is oral.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrastemal injection, and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a ligand, drug, or other materialother than directly into the central nervous system, such that it entersthe patient's system and thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

The PEG-carfilzomib compounds of the invention described herein may beadministered to humans and other animals for therapy by any suitableroute of administration, including orally, nasally, as by, for example,a spray, rectally, intravaginally, parenterally, intracistemally, andtopically, as by powders, ointments or drops, including buccally andsublingually.

Regardless of the route of administration selected, a compound of theinvention, which may be used in a suitable hydrated form, and/or thepharmaceutical compositions provided herein, is formulated into apharmaceutically acceptable dosage form by conventional methods known tothose of skill in the art.

As mentioned herein above, actual dosage amounts of the PEG carfilzomibcompound in the pharmaceutical compositions provided by the inventionmay be varied so as to include an amount of the active PEG-freecarfilzomib agent which is clinically proven to be effective, and/orcommercially approved as effective, to achieve the desired therapeuticresponse for a cancer patient, including without limitation, for amultiple myeloma patient. To this end, the specific amount of and/orconcentration of a pegylated carfilzomib compound of the invention in apharmaceutically acceptable composition will vary depending on severalfactors, including the dosage of the compound to be administered, thepharmacokinetic characteristics of the compound(s) employed, and theroute of administration. In general, the pharmaceutical compositionsprovided by the invention may be an aqueous solution containing about0.1-20% w/v of a compound disclosed herein, among other substances, forparenteral administration. Typical dose ranges for the PEG carfilzomibcompound active ingredients are from about 0.01 to about 50 mg/kg ofbody weight per day, given in 1-4 divided doses each day. Each divideddose will contain one or more of the compounds provided by theinvention. The desired, specific compound dosage amount should be anamount sufficient to provide a therapeutically effective dosage of freeacting carfilzomib in the plasma of the patient, the effective dosageamount being based on regulatory approved use, for regulatory approvedindications. This effective amount may vary from patient to patient, andis generally dependent on several factors including the overall healthof a patient, and the specific formulation composition and route ofadministration of the chosen compound(s).

Carfilzomib is currently approved in doses, provided once daily for thefirst 2 consecutive days every week for 3 consecutive weeks in a 28 daycycle, in an amount sufficient to provide a patient plasma concentrationranging from 20 mg/m² to 56 mg/m². Thus, a higher molecule weight PEGcarfilzomib compound of the invention should be administered in amountssufficient to pharmacokinetically provide amounts approximatelyequivalent to approved dosing ranges. For example a 2K PEG compound ofthe invention is approximately 24% by weight of free carfilzomib. Thus,using an average male with 1.9 m2 average body surface, to achieve aboutan equivalent dose of 27 mg/m², one would have to dose about 215 mg ofthe 2 k PEG CFZ compound. Similarly, one may dose about 1100 mg of a 20KPEG CFZ compound to deliver the same amount of carfilzomib as would a 70mg/m² dose of the currently approved formulation for carfilzomib.

In embodiment 71 of the invention, there is provided a method oftreating cancer in a subject in need of treatment, the method comprisingadministering to the subject an effective dosage amount of a PEGcarfilzomib compound of Formula I to the subject to treat the cancer. Inembodiment 72, the invention provides the method of embodiment 71wherein the cancer is multiple myeloma. In embodiment 73, the inventionprovides the method of any one of embodiments 71-72 wherein theeffective dosage amount of PEG carfilzomib is in the range from about100 mg to about 2000 mg. In embodiment 74, the invention provides themethod of any one of embodiments 71-73 wherein the effective dosageamount is in the range from about 150 mg to about 1000 mg per day. Inembodiment 75, the invention provides the method of any one ofembodiments 71-74 wherein the effective dosage amount of the PEGcarfilzomib compound administered is in the range from about 200 mg toabout 500 mg per day. In embodiment 76, the invention provides themethod of any one of embodiments 71-73 wherein the effective dosageamount of a 2K PEG carfilzomib compound administered is in the rangefrom about 150 mg to about 600 mg per day. In embodiment 77, theinvention provides the method of any one of embodiments 71-73 whereinthe effective dosage amount of a 3K PEG carfilzomib compoundadministered is in the range from about 300 mg to about 2000 mg per day.In embodiment 78, the invention provides the method of any one ofembodiments 71-73 wherein the effective dosage amount of a 5K PEGcarfilzomib compound administered is in the range from about 800 mg toabout 3000 mg per day. In embodiment 79, the invention provides themethod of any one of embodiments 71-73 wherein the effective dosageamount of a 20K PEG carfilzomib compound administered is in the rangefrom about 800 mg to about 3000 mg per day. In embodiment 80, theinvention provides the method of any one of embodiments 71-73 whereinthe effective dosage amount of a PEG carfilzomib compound administeredis in the range from about 200 mg to about 1500 mg per day. Inembodiment 81, the invention provides the method of any one ofembodiments 71-73 wherein the effective dosage amount of the PEGcarfilzomib compound administered is in the range from about 5 mg/kg toabout 50 mg/kg by weight of the subject per day. In embodiment 82, theinvention provides the method of any one of embodiments 71-73 whereinthe effective dosage amount of a 2K, 3K or 5K PEG carfilzomib compoundadministered is in the range from about 200 mg to about 800 mg per day.In embodiment 83, the invention provides the method of any one ofembodiments 71-73 wherein the effective dosage amount of a 2K or 3K PEGcarfilzomib compound administered is in the range from about 200 mg toabout 500 mg per day. In embodiment 84, the invention provides themethod of any one of embodiments 71-73 wherein the effective dosageamount of a 5K or 20K PEG carfilzomib compound administered is in therange from about 400 mg to about 1000 mg per day. In embodiment 85, theinvention provides the method of any one of embodiments 71-84, whereinthe method further comprises administration of a steroid. In embodiment86, the invention provides the method of embodiment 85 wherein thesteroid is selected from the group consisting of dexamethasone andprednisone. In embodiment 87, the invention provides the method of anyone of embodiments 85-86 wherein the steroid is dexamethasone. Inembodiment 88, the invention provides the method of any one ofembodiment 85-86 wherein the steroid is prednisone. In embodiment 89,the invention provides the method of any one of embodiments 71-88wherein the method further comprises administration of animmunomodulatory agent selected from the group consisting ofthalidomide, lenalidomide and pomalidomide. In embodiment 90, theinvention provides the method of embodiment 89, wherein theimmunomodulatory agent is lenalidomide or pomalidomide. In embodiment91, the invention provides the method of any one of embodiments 89-90,wherein the immunomodulatory agent is lenalidomide. In embodiment 92,the invention provides the method of any one of embodiments 89-90,wherein the immunomodulatory agent is pomalidomide. In embodiment 93,the invention provides the method of any one of embodiments 71-88wherein the method further comprises administration of a CD-38inhibiting agent. In embodiment 94, the invention provides the method ofembodiment 93, wherein the CD-38 inhibiting agent is daratumumab. Inembodiment 95, the invention provides the method of any one ofembodiments 71-94 wherein the cancer is relapsed or refractory multiplemyeloma. In embodiment 96, the invention provides the method of any oneof embodiments 71-94 wherein the cancer is new diagnosed multiplemyeloma. In embodiment 97, the invention provides the method ofembodiment 96 wherein the cancer is new diagnosed multiple myeloma andwherein the patient is stem cell transplant eligible, as determined by alicensed, authorized medical practitioner. In embodiment 98, theinvention provides the method of embodiment 96 wherein the cancer is newdiagnosed multiple myeloma and wherein the patient is not stem celltransplant eligible, as determined by a licensed, authorized medicalpractitioner. In embodiment 99, the invention provides the method of anyone of embodiments 71-98, wherein the method comprises administering tothe subject a pharmaceutical composition comprising a PEG carfilzomibcompound of Formula I. In embodiment 100, the invention provides themethod of embodiment 99 wherein the pharmaceutical composition is anoral solution or a parenteral solution. In embodiment 101, the inventionprovides the method of any one of embodiments 99-100 wherein thepharmaceutical composition is a freeze-dried preparation that can bereconstituted prior to administration. In embodiment 102, the inventionprovides the methods of each one of embodiments 71-98, wherein the PEGcarfilzomib compound is

wherein R¹ is C₁₋₁₀alkyl;

R² is C₁₋₆alkyl, —OCH₃ or halogen;

R³ is H or CH₃;

X⁻ is a counter anion selected from chloride anion and a alkyl-sulfonateanion;

n is 4; and

PEG is a polyethylene glycol polymeric moiety of 3000, 5000 or 20000dalton molecular weight.

In embodiment 103, the invention provides the methods of embodiment71-98, wherein the PEG carfilzomib compound is

In embodiment 104, the invention provides the methods of embodiment71-98, wherein the PEG carfilzomib compound is

Combinations

While a PEG-carfilzomib compound of the invention can be dosed oradministered as the sole active pharmaceutical agent, it can also beused in combination with one or more agents, such a second anti-canceragent. When administered as a combination, the PEG carfilzomib activeingredient and the other agent may be formulated as separatecompositions that are administered simultaneously or sequentially atdifferent times, or both active agents can be given as a singlecomposition.

The phrase “co-therapy” (or “combination-therapy”), in defining the useof PEG carfilzomib compound of the present invention and anotheranti-cancer agent, is intended to embrace administration of each agentin a sequential manner in a regimen that will provide beneficial effectsof the drug combination, and is intended as well to embraceco-administration of these agents in a substantially simultaneousmanner, such as in a single dosage formulation having a fixed ratio ofthese active agents, or in multiple, separate dosage formulations foreach active agent. Thus, the invention is not limited in the sequence ofadministration, i.e, the PEG carfilzomib compound(s) may be administeredeither prior to, simultaneous with or after administration of the otheragent.

In certain embodiments, a PEG-carfilzomib compound described herein isconjointly administered with one or more other proteasome inhibitor(s).Another proteasome inhibitor may include, for example, bortezomib,oprozomib or ixazomib. In another embodiment, the PEG carfilzomibcompound described herein is administered in combination with animmunomodulatory compound, including thalidomide, lenalidomide andpomalidomide. In an embodiment from the immediately precedingembodiment, the PEG carfilzomib is administered in combination with animmunomodulatory agent selected from lenalidomide and pomalidomide. In afurther embodiment, the invention provides a method of treating cancerin a subject by administering to the subject a combination therapycomprising a PEG carfilzomib compound of Formula I or II and animmunomodulatory agent. In a further embodiment, the cancer is multiplemyeloma.

In certain embodiments, a PEG-carfilzomib compound described herein isconjointly administered with one or more chemotherapeutics. Suitablechemotherapeutics may include, natural products such as vinca alkaloids(i.e. vinblastine, vincristine, and vinorelbine), taxanes (e.g.,docetaxel, paclitaxel, e.g., docetaxel), epidipodophyllotoxins (i.e.etoposide, teniposide), antibiotics (dactinomycin (actinomycin D)daunorubicin, doxorubicin and idarubicin; e.g., doxorubicin),anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin) andmitomycin, enzymes (L-asparaginase which systemically metabolizesL-asparagine and deprives cells which do not have the capacity tosynthesize their own asparagine); antiplatelet agents;antiproliferative/antimitotic alkylating agents such as nitrogenmustards (mechlorethamine, ifosphamide, cyclophosphamide and analogs,melphalan, chlorambucil, e.g., melphalan), ethylenimines andmethylmelamines (hexaamethylmelaamine and thiotepa), alkyl sulfonates(busulfan), nitrosoureas (carmustine (BCNU) and analogs, streptozocin),trazenes-dacarbazinine (DTIC); antiproliferative/antimitoticantimetabolites such as folic acid analogs (methotrexate), pyrimidineanalogs (fluorouracil, floxuridine, and cytarabine), purine analogs andrelated inhibitors (mercaptopurine, thioguanine, pentostatin and2-chlorodeoxyadenosine); aromatase inhibitors (anastrozole, exemestane,and letrozole); platinum coordination complexes (cisplatin,carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide;DNA binding/Cytotoxic agents (e.g., Zalypsis); histone deacetylase(HDAC) inhibitors (e.g., trichostatin, sodium butyrate, apicidan,suberoyl anilide hydroamic acid (SAHA (Vorinostat)), trichostatin A,depsipeptide, apicidin, A-161906, scriptaid, PXD-101, CHAP, butyricacid, depudecin, oxamflatin, phenylbutyrate, valproic acid, MS275(N-(2-Aminophenyl)-4-[N-(pyridine-3-ylmethoxy-carbonyl)aminomethyl]benzamide),LAQ824/LBH589, CI994, MGCD0103, ACY-1215, Panobinostat); hormones (i.e.estrogen) and hormone agonists such as leutinizing hormone releasinghormone (LHRH) agonists (goserelin, leuprolide and triptorelin). Otherchemotherapeutic agents may include mechlorethamine, camptothecin,ifosfamide, tamoxifen, raloxifene, gemcitabine, navelbine, or any analogor derivative variant of the foregoing.

In certain embodiments, a PEG-carfilzomib compound described herein isconjointly administered with a cytokine. Cytokines include, but are notlimited to, Interferon-γ, -α, and -β, Interleukins 1-8, 10 and 12,Granulocyte Monocyte Colony-Stimulating factor (GM-CSF), TNF-α and -β,and TGF-β.

In certain embodiments, a PEG-carfilzomib compound described herein isconjointly administered with a steroid. Suitable steroids may include,but are not limited to, 21-acetoxypregnenolone, alclometasone,algestone, amcinonide, beclomethasone, betamethasone, budesonide,chloroprednisone, clobetasol, clocortolone, cloprednol, corticosterone,cortisone, cortivazol, deflazacort, desonide, desoximetasone,dexamethasone, diflorasone, diflucortolone, difuprednate, enoxolone,fluazacort, flucloronide, flumethasone, flunisolide, fluocinoloneacetonide, fluocinonide, fluocortin butyl, fluocortolone,fluorometholone, fluperolone acetate, fluprednidene acetate,fluprednisolone, flurandrenolide, fluticasone propionate, formocortal,halcinonide, halobetasol propionate, halometasone, hydrocortisone,loteprednol etabonate, mazipredone, medrysone, meprednisone,methylprednisolone, mometasone furoate, paramethasone, prednicarbate,prednisolone, prednisolone 25-diethylaminoacetate, prednisolone sodiumphosphate, prednisone, prednival, prednylidene, rimexolone, tixocortol,triamcinolone, triamcinolone acetonide, triamcinolone benetonide,triamcinolone hexacetonide, and salts and/or derivatives thereof (e.g.,hydrocortisone, dexamethasone, methylprednisolone and prednisolone;e.g., dexamethasone). In certain embodiments, a PEG-carfilzomib compounddescribed herein are conjointly administered with dexamethasone. Incertain embodiments, conjoint therapy includes the dosing regimensprovided on the KYPROLIS (carfilzomib) label, as approved by the US FDAand by the EMA.

In some embodiments, a PEG-carfilzomib compound described herein isconjointly administered with an immunotherapeutic agent. Suitableimmunotherapeutic agents may include, but are not limited to, MDRmodulators (verapamil, valspordar, biricodar, tariquidar, laniquidar),cyclosporine, thalidomide, and monoclonal antibodies. The monoclonalantibodies can be either naked or conjugated such as rituximab,tositumomab, alemtuzumab, epratuzumab, ibritumomab tiuxetan, gemtuzumabozogamicin, bevacizumab, cetuximab, erlotinib and trastuzumab.

In certain embodiments, a PEG-carfilzomib compound described herein isconjointly administered with one or more histone deacetylase (HDAC)inhibitors (e.g., trichostatin, sodium butyrate, apicidan, suberoylanilide hydroamic acid (“SAHA” (Vorinostat)), trichostatin A,depsipeptide, apicidin, A-161906, scriptaid, PXD-101, CHAP, butyricacid, depudecin, oxamflatin, phenylbutyrate, valproic acid, MS275(N-(2-Aminophenyl)-4-[N-(pyridine-3-ylmethoxy-carbonyl)aminomethyl]benzamide),LAQ824/LBH589, CI994, MGCD0103, ACY-1215, Panobinostat; e.g., SAHA,ACY-1215, Panobinostat).

In certain embodiments, a PEG-carfilzomib compound described herein isconjointly administered with one or more nitrogen mustards(mechlorethamine, ifosphamide, cyclophosphamide and analogs, melphalan,chlorambucil, e.g., melphalan). In certain embodiments, aPEG-carfilzomib compound described herein is conjointly administeredwith one or more DNA binding/Cytotoxic agents (e.g., Zalypsis). Incertain embodiments, a PEG-carfilzomib compound described herein isconjointly administered with one or more taxanes (e.g., docetaxel,paclitaxel, e.g., docetaxel).

In certain embodiments, a PEG-carfilzomib compound described herein isconjointly administered with one or more antibiotics (dactinomycin(actinomycin D) daunorubicin, doxorubicin and idarubicin; e.g.,doxorubicin).

The foregoing is merely illustrative of the invention and is notintended to limit the invention to the disclosed uses. Variations andchanges, which are routine to one skilled in the art, are intended to bewithin the scope and nature of the invention, which are defined in theappended claims.

What is claimed is:
 1. A pegylated carfilzomib compound having astructure of formula I

or a pharmaceutically acceptable salt thereof, wherein R¹ is C₁₋₁₀alkylor C₃₋₇ cycloalkyl; each R², independently, is C₁₋₆ alkyl, —OCH₃ orhalogen; o is an integer selected from 0, 1, 2 or 3; linker is a moietyhaving the structure of

wherein R³ is H or CH₃; n is an integer selected from 1, 2, 3 or 4; p isan integer selected from 0, 1, 2, 3 or 4; q is an integer selected from1, 2, 3, 4, 5, 6, 7, 8 or 9; r is an integer selected from 0, 1, 2, 3, 4or 5; and PEG is a polyethylene glycol polymeric moiety having amolecular weight ranging from about 500 to about 20,000.
 2. The compoundof claim 1 wherein R¹ is C₁₋₁₀alkyl.
 3. The compound of claim 1 whereineach o is 0 or 1 and R² is CH₃ or halogen.
 4. The compound of claim 1wherein each o is 0 or 1 and R² is CH₃ or F.
 5. The compound of claim 1wherein the linker is a moiety having the structure of

wherein R³ is H or CH₃; q is an integer selected from 1, 2, 3, 4 or 5;and r is an integer selected from 0, 1, 2, 3 or
 4. 6. The compound ofclaim 1 wherein the linker is

wherein R³ is H or CH₃; q is 4; and r is
 2. 7. The compound of claim 2wherein R¹ is methyl, ethyl, propyl, isopropyl, butyl, t-butyl, pentyl,hexyl or heptyl.
 8. The compound of claim 1 wherein R¹ is methyl, ethyl,propyl, isopropyl, butyl, t-butyl, pentyl, hexyl or heptyl; and thelinker is


9. The pegylated carfilzomib compound of claim 1 having a structure ofFormula II

wherein R¹ is C₁₋₁₀alkyl or C₃₋₇cycloalkyl; R² is C₁₋₆alkyl, —OCH₃ orhalogen; linker is a moiety having the structure of

wherein R³ is H or CH₃; n is an integer selected from 1, 2, 3 or 4; p isan integer selected from 0, 1, 2, 3 or 4; q is an integer selected from1, 2, 3, 4, 5, 6, 7, 8 or 9; r is an integer selected from 0, 1, 2, 3, 4or 5; X is a counter ion salt selected from a chloride, a bisulfate, asulfate, a nitrate, a phosphate, an alky-sulfonate or an aryl-sulfonate;and PEG is a polyethylene glycol polymeric moiety having a molecularweight ranging from about 2000 to about 20,000.
 10. The compound ofclaim 9 wherein R¹ is C₁₋₁₀alkyl.
 11. The compound of claim 9 wherein R²is H, CH₃ or halogen.
 12. The compound of claim 9 wherein R² is H, CH₃or F.
 13. The compound of claim 9 wherein the linker is a moiety havingthe structure of

wherein R³ is H or CH₃; q is an integer selected from 1, 2, 3, 4 or 5;and r is an integer selected from 0, 1, 2, 3 or
 4. 14. The compound ofclaim 9 wherein the linker is

wherein R³ is H or CH₃; q is 4; and r is
 2. 15. The compound of claim 9wherein R¹ is methyl, ethyl, propyl, isopropyl, butyl, t-butyl, pentyl,hexyl or heptyl.
 16. The compound of claim 9 wherein R¹ is methyl,ethyl, propyl, isopropyl, butyl, t-butyl, pentyl, hexyl or heptyl; andthe linker is


17. The compound of claim 1 having the structure of


18. The compound of claim 1 wherein the PEG has a weight ranging fromabout 2K to about 20K.
 19. The compound of claim 1 wherein the PEG has aweight of 2K, 3K, 5K or 20K.
 20. The compound of claim 1 that is apharmaceutically acceptable salt comprising a counter anion selectedfrom a chloride anion, a bisulfate anion, a sulfate anion, a nitrateanion, a phosphate anion, an alky-sulfonate anion or an aryl-sulfonateanion.
 21. The compound of claim 20 wherein the counter anion is achloride anion or an alky-sulfonate anion.
 22. A pharmaceuticalcomposition comprising the compound according to claim 1 and apharmaceutically acceptable excipient, carrier or diluent.
 23. Thepharmaceutical composition of claim 22 that is administered orally or byinfusion or injection.
 24. A method of treating multiple myelomacomprising administering to a patient in need thereof, a therapeuticallyeffective amount of the compound of claim for a composition of claim 22.25. The method of claim 24 wherein the multiple myeloma is relapsed,refractory or relapsed and refractory multiple myeloma.
 26. The methodof claim 24 wherein the multiple myeloma is newly diagnosed multiplemyeloma.
 27. A process of making the compound according to claim 1, theprocess comprising the step of

wherein X− is a counter ion salt selected from the group consisting of achloride anion, a bisulfate anion, a sulfate anion, a nitrate anion, aphosphate anion, an alky-sulfonate anion or an aryl-sulfonate anion, andPEG has a weight ranging from about 2K to about 20K, to prepare acompound of Formula I